Vectors for delivery of light sensitive proteins and methods of use

ABSTRACT

Provided herein are compositions and methods for gene and etiology-nonspecific and circuit-specific treatment of diseases, utilizing vectors for delivery of light-sensitive proteins to diseased and normal cells and tissues of interest.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application Nos.61/054,571 filed May 20, 2008, 61/199,241 filed Nov. 14, 2008, and61/200,430 filed Nov. 26, 2008, which applications are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

A gene-delivery therapy to treat a disease or disorder independent oftreating an underlying mutation could have potential value. Methodscapable of controlling, regulating, and/or driving specific neuralcircuits so as to mediate naturalistic neural responses and highresolution perception and control could also be of enormous potentialtherapeutic value. Neurons are an example of a type of cell that usesthe electrical currents created by depolarization to generatecommunication signals (e.g., nerve impulses). Other electricallyexcitable cells include skeletal muscle, cardiac muscle, and endocrinecells. Neurons use rapid depolarization to transmit signals throughoutthe body and for various purposes, such as motor control (e.g., musclecontractions), sensory responses (e.g., touch, hearing, and othersenses) and computational functions (e.g., brain functions). Byfacilitating or inhibiting the flow of positive or negative ions throughcell membranes, the cell can be briefly depolarized, depolarized andmaintained in that state, or hyperpolarized. Thus, the control of thedepolarization of cells can be beneficial for a number of differentpurposes, including visual, muscular and sensory control.Light-sensitive protein channels, pumps, and receptors can permitmillisecond-precision optical control of cells. Although light-sensitiveproteins in combination with light can be used to control the flow ofions through cell membranes, targeting and delivery remain to beaddressed for specific diseases, disorders, and circuits.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a recombinant nucleic acidcomprising a nucleic acid encoding a light-sensitive protein operativelylinked to a metabotropic glutamate receptor 6 (mGluR6) regulatorysequence or fragment thereof. In one embodiment the light-sensitiveprotein can be selected from the group consisting of ChR1, ChR2, VChR1,ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD,ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants thereof. Inanother embodiment the light-sensitive protein is ChR2 or alight-sensitive protein that is at least about 70%, at least about 80%,at least about 90% or at least about 95% identical to ChR2. In anotherembodiment the mGluR6 regulatory sequence fragment comprises less thanabout 1000, less than about 750, less than about 500, less than about250, or less than about 100 base pairs. In a related embodiment themGluR6 regulatory sequence or fragment thereof is a mGluR6 promoter orenhancer. In a specific related embodiment the nucleic acid furthercomprises a green fluorescent protein. In another embodiment the nucleicacid is encapsidated within a recombinant adeno-associated virus (AAV).In certain embodiments, the recombinant AAV is of a combinatorial hybridof 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more serotypes ormutants thereof. In certain embodiments, the recombinantadeno-associated virus is of a serotype selected from the groupconsisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAV10, AAV11, AAV12, and hybrids thereof. In a related embodiment, thenucleic acid is encapsidated within a recombinant virus selected fromthe group consisting of recombinant adeno-associated virus (AAV),recombinant retrovirus, recombinant lentivirus, and recombinantpoxvirus.

In another aspect the invention provides a vector comprising a nucleicacid encoding a light-sensitive protein, said nucleic acid operativelylinked to a metabotropic glutamate receptor 6 (mGluR6) regulatorysequence or fragment thereof. In one embodiment the light-sensitiveprotein is selected from the group consisting of ChR1, ChR2, VChR1, ChR2C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF,ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants thereof. In a relateembodiment the light-sensitive protein is ChR2 or a light-sensitiveprotein that is at least about 70%, at least about 80%, at least about90% or at least about 95% identical to ChR2. In another embodiment themGluR6 regulatory sequence fragment is less than about 1000, less thanabout 750, less than about 500, less than about 250, or less than about100 base pairs. In a related embodiment the mGluR6 regulatory sequencefragment is represented by the sequence in FIG. 6. In another embodimentthe vector comprises a recombinant adeno-associated virus (AAV). In arelated embodiment the vector comprises a recombinant virus selectedfrom the group consisting of recombinant adeno-associated virus (AAV),recombinant retrovirus, recombinant lentivirus, and recombinantpoxvirus. In a specific embodiment the AAV is of a serotype selectedfrom the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof. In other specificembodiments, the recombinant AAV is of a combinatorial hybrid of 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more serotypes or mutantsthereof. In a related embodiment the AAV comprises mutated capsidprotein. In one specific embodiment the capsid protein comprises amutated tyrosine residue. The mutated tyrosine residue can be selectedfrom the group consisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F,Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and Y720F. In a specificembodiment the mutated capsid protein comprises a tyrosine residuemutated to a phenylalanine.

In another aspect the present invention provides a method of treating asubject suffering from a disease or disorder of the eye comprisingintroducing into an affected eye a recombinant adeno-associated virus(AAV) comprising a light-sensitive protein operatively linked to ametabotropic glutamate receptor 6 regulatory sequence (mGluR6) orfragment thereof. In one embodiment the disease or disorder of the eyeis caused by photoreceptor cell degeneration. In another embodiment thelight-sensitive protein is selected from the group consisting of ChR1,ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, andvariants thereof. In a related embodiment the light-sensitive protein isChR2 or a light-sensitive protein that is at least about 70%, at leastabout 80%, at least about 90% or at least about 95% identical to ChR2.In another embodiment the mGluR6 promoter fragment is less than about1000, less than about 750, less than about 500, less than about 250, orless than about 100 base pairs. In a related embodiment the mGluR6promoter fragment is represented by the sequence in FIG. 6. In anotherembodiment the AAV is of a serotype selected from the group consistingof AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,AAV12, and hybrids thereof. In other embodiments, the recombinant AAV isof a combinatorial hybrid of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15 or more serotypes or mutants thereof. In a related embodiment the AAVcomprises a mutated capsid protein. In another related embodiment thecapsid protein comprises a mutated tyrosine residue. In a specificembodiment the mutated tyrosine residue is selected from the groupconsisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F,Y281F, Y508F, Y576F, Y612G, Y673F and Y720F. In a related embodiment themutated capsid protein comprises a tyrosine residue mutated to aphenylalanine. In another embodiment the AAV is introduced usingintravitreal injection, subretinal injection and/or ILM peel. In aspecific embodiment the AAV is introduced into a retinal bipolar cell(e.g. ON or OFF retinal bipolar cells; rod and cone bipolar cells). Inanother embodiment the method further comprises using a light-generatingdevice external to the eye.

In another aspect the present invention provides a method of expressingan exogenous nucleic acid in a retinal bipolar cell (e.g. ON or OFFretinal bipolar cells; rod and cone bipolar cells) comprisingintroducing into a retina a vector comprising the exogenous nucleicoperatively linked to a retinal bipolar (e.g. ON or OFF retinal bipolarcells; rod and cone bipolar cells) cell-specific regulatory sequencewherein the method results in at least about a 25-30% transductionefficiency. In other embodiment, such method results in at least about a10% transduction efficiency. In one embodiment the method results in atleast about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% transductionefficiency. In a related embodiment the transduction efficiency ismeasured by quantifying the total number of retinal bipolar cells (e.g.ON or OFF retinal bipolar cells; rod and cone bipolar cells) infected.In another embodiment the exogenous nucleic acid comprises alight-sensitive protein. In a related embodiment the light-sensitiveprotein is selected from the group consisting of ChR1, ChR2, VChR1, ChR2C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF,ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants thereof. In a specificembodiment the light-sensitive protein is ChR2 or a light-sensitiveprotein that is at least about 70%, at least about 80%, at least about90% or at least about 95% identical to ChR2. In another embodiment theregulatory sequence comprises a metabotropic glutamate receptor 6regulatory sequence (mGluR6) or a fragment thereof. In a relatedembodiment the mGluR6 regulatory sequence fragment is less than about1000, less than about 750, less than about 500, less than about 250, orless than about 100 base pairs. In a specific embodiment the mGluR6regulatory sequence fragment is represented by the sequence in FIG. 6.In another embodiment the exogenous nucleic acid is introduced using arecombinant adeno-associated viral vector (AAV). In a related embodimentthe AAV comprises a mutated capsid protein. In a specific embodiment thecapsid protein comprises a mutated tyrosine residue. In a relatedembodiment the mutated tyrosine residue is selected from the groupconsisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F,Y281F, Y508F, Y576F, Y612G, Y673F and Y720F. In another embodiment themutated capsid protein comprises a tyrosine residue mutated to aphenylalanine. In another embodiment the exogenous nucleic acid isintroduced using intravitreal injection, subretinal injection, and/orILM peel. In another embodiment the AAV is of a serotype is selectedfrom the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof. In yet anotherembodiments, the recombinant AAV is of a combinatorial hybrid of 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more serotypes or mutantsthereof.

In yet another aspect, the present invention provides a method ofintroducing an exogenous nucleic acid into the nucleus of a retinal cellcomprising introducing a vector comprising an exogenous nucleic acidoperatively linked to a retinal cell-specific regulatory sequence into aretinal cell, wherein the vector is specifically designed to avoidubiquitin-mediated protein degradation. In one embodiment thedegradation is proteasome-mediated. In another embodiment the exogenousnucleic acid comprises a light-sensitive protein. In a relatedembodiment the light-sensitive protein is selected from the groupconsisting of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T,ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR,melanopsin, and variants thereof. In another related embodiment thelight-sensitive protein is ChR2 or a light-sensitive protein that is atleast about 70%, at least about 80%, at least about 90% or at leastabout 95% identical to ChR2. In another embodiment the retinal cell is aretinal bipolar cell (e.g. ON or OFF retinal bipolar cells; rod and conebipolar cells). In a related embodiment the regulatory sequencecomprises a metabotropic glutamate receptor 6 promoter (mGluR6 promoter)or fragment thereof. In another embodiment the mGluR6 fragment is lessthan 1000, 750, 500, 250, or 100 base pairs. In another embodiment themGluR6 promoter fragment is represented by the sequence in FIG. 6. Inanother embodiment the vector is selected from the group consisting ofrecombinant adeno-associated virus (AAV), recombinant retrovirus,recombinant lentivirus, and recombinant poxvirus. In a relatedembodiment the vector is a recombinant adeno-associated viral vector(AAV). In another embodiment the AAV is of a serotype selected from thegroup consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, AAV12, and hybrids thereof. In other embodiments,the recombinant AAV is of a combinatorial hybrid of 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 or more serotypes or mutants thereof. Inanother embodiment the AAV comprises a mutated capsid protein. Inanother embodiment the capsid protein comprises a mutated tyrosineresidue. In another embodiment the mutated tyrosine residue is selectedfrom the group consisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F,Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and Y720F. In anotherembodiment the mutated capsid protein comprises a tyrosine residuemutated to a phenylalanine. In another embodiment the vector isintroduced using intravitreal injection, subretinal injection, and/orILM peel.

In another aspect the present invention provides a method of transducinga retinal bipolar cell (e.g. ON or OFF retinal bipolar cells; rod andcone bipolar cells) comprising introducing into a retina a vectorcomprising an exogenous nucleic acid operatively linked to a regulatorysequence. In one embodiment the regulatory sequence is a non-cell typespecific promoter. In another embodiment the regulatory sequence is aguanine nucleotide binding protein alpha activating activity polypeptideO (GNAO1) promoter or a fusion of the cytomegalovirus (CMV) immediateearly enhancer and the bovine β-actin promoter plus intron1-exon1junction (CBA, smCBA). In another embodiment the exogenous nucleic acidcomprises a light-sensitive protein. In a related embodiment thelight-sensitive protein is selected from the group consisting of ChR1,ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, andvariants thereof. In another related embodiment the light-sensitiveprotein is ChR2 or a light-sensitive protein that is at least about 70%,at least about 80%, at least about 90% or at least about 95% identicalto ChR2. In another embodiment the vector is selected from the groupconsisting of recombinant adeno-associated virus (AAV), recombinantretrovirus, recombinant lentivirus, and recombinant poxvirus. In arelated embodiment the vector is a recombinant adeno-associated viralvector (AAV). In another embodiment the AAV is of a serotype selectedfrom the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof. In certainembodiments, the recombinant AAV is of a combinatorial hybrid of 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more serotypes or mutantsthereof. In a related embodiment the AAV comprises a mutated capsidprotein. In another embodiment the capsid protein comprises a mutatedtyrosine residue. In another embodiment the mutated tyrosine residue isselected from the group consisting of Y252F, Y272F, Y444F, Y500F, Y700F,Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and Y720F. Inanother embodiment the mutated capsid protein comprises a tyrosineresidue mutated to a phenylalanine. In another embodiment the vector isintroduced using intravitreal injection, subretinal injection, and/orILM peel.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts the ChR1 nucleic acid sequence.

FIG. 2 depicts the ChR2 nucleic acid sequence.

FIG. 3 depicts the NpHR nucleic acid sequence.

FIG. 4 depicts the melanopsin nucleic acid sequence.

FIG. 5 depicts the ChR2 nucleic acid sequence that is mammaliancodon-optimized and that encodes a ChR2 fused with Green FluorescentProtein (GFP).

FIG. 6 depicts a fragment of the GRM6 (metabotropic glutamate receptor6) regulatory nucleic acid sequence capable of regulating expression ina bipolar cell specific manner.

FIG. 7 depicts a smCBA regulatory nucleic acid sequence.

FIG. 8 depicts: neurons expressing ChR2 and firing. (A) Neuronsexpressing ChR2 with light stimulation; (B) Neurons firing in responseto fast trains of blue light pulses.

FIG. 9 depicts: AAV Delivery to retinal bipolar cells. Column 1 showsGFP expression in retinal bipolar cells after a subretinal injectionwith the AAV7-CBA-GFP vector after 8 weeks of age. Column 2 shows PKCαstaining (an antibody that is specific to bipolar cells), column 3 showsDAPI staining for cell nuclei, and column 4 shows merged images of GFPexpression, PKCα and DAPI stains. The first row is 20× magnification andthe second row is 40× magnification.

FIG. 10 depicts: Expression of the ChR2-GFP fused protein in rd1, rho−/−, and rd16 in retinal bipolar cells. In each image, the retinalpigment epithelium (RPE), bipolar cells or inner nuclear layer (INL),inner plexiform layer (IPL), and ganglion cell layer (GCL) are noted.The brighter white areas show GFP expression. There are ringlets ofexpression in the bipolar cells of the INL (except for the AAV5intravitreal injection).

FIG. 11 depicts: analysis of EGFP expression in frozen retinal sectionsby immunohistochemistry at 1 month following subretinal injections withthe Tyrosine mutant AAV vectors. Example sections depicting spread andintensity of EGFP fluorescence throughout the retina after transductionwith serotype 2 Y444 (a) or serotype 8 Y733 (b). The images are orientedwith the vitreous toward the bottom and the photoreceptor layer towardthe top. EGFP fluorescence in photoreceptors, RPE and ganglion cellsfrom mouse eyes injected subretinally with serotype 2 Y444 (c) EGFPfluorescence in photoreceptors, RPE and Müller cells after serotype 8Y733 delivery (d) Detection of Müller cells processes (red) byimmunostaining with a glutamine-synthetase (GS) antibody (e) Mergedimage showing colocalization of EGFP fluorescence (green) and GSstaining (red) in retinal sections from eyes treated with serotype 8Y733 (f) Calibration bar 100 μM. gcl, ganglion cell layer; ipl, innerplexiform layer; inl, inner nuclear layer; onl, outer nuclear; os, outersegment; rpe, retinal pigment epithelium.

FIG. 12 depicts: Training mice on a water maze task. (A) A schematic ofthe water maze used to measure scotopic threshold (Hayes and Balkema,1993). (B) Time it took each mouse group (retinal degenerated—treated,retinal degenerated—untreated, and wild type) to find the target (blackplatform+LED array) as a function of training sessions. (C) Time it tookeach mouse group (treated rd1, treated rd16, treated rho −/−, untreatedretinal degenerated, and wild type) to find the target (blackplatform+LED array) as a function of different light intensities.

FIG. 13 depicts: goggle-like device with an associated lightgeneration/production element (LED array/laser system) that can triggerexpression of light-sensitive proteins.

DETAILED DESCRIPTION OF THE INVENTION Light-Sensitive Proteins

The present invention provides recombinant nucleic acids encodinglight-sensitive proteins, viral and non-viral vectors for the deliveryof recombinant nucleic acids encoding light-sensitive proteins, andmethods for delivery of light-sensitive proteins.

Light-sensitive proteins are proteins that belong to the opsin familyand include vertebrate (animal) and invertebrate rhodopsins. The animalopsins, rhodopsins, are G-protein coupled receptors (GPCRs) with7-transmembrane helices which can regulate the activity of ion channels.Invertebrate rhodopsins are usually not GPCRs, but are light-sensitiveor light-activated ion pumps or ion channels.

An algal opsin such as channelrhodopsin (ChR2) from Chlamydomonasreinhardtii allows blue light-induced action potentials to be triggeredwith millisecond-precision in cells due to depolarizing cation fluxthrough a light-gated pore. An archael opsin such as halorhodopsinNatronomonas pharaonis allows for light-activated chloride pumping; thepump can be hyperpolarized and inhibited from firing action potentialswhen exposed to yellow light. Use of such light-sensitive opsins allowsfor temporal and spatial regulation of neuronal firing activity.

As referred to herein, a “light-sensitive” protein includeschannelrhodopsins (ChR1, ChR2), halorhodopsins (NpHR), melanopsins,pineal opsins, bacteriorhodopsin, and variants thereof. Alight-sensitive protein of this invention can occur naturally in plant,animal, archaebacterial, algal, or bacterial cells, or can alternativelybe created through laboratory techniques.

Channelrhodopsins ChR1 (GenBank accession number AB058890/AF385748;FIG. 1) and ChR2 (GenBank accession number AB058891/AF461397; FIG. 2)are two rhodopsins from the green alga Chlamydomonas reinhardtii (Nagel,2002; Nagel, 2003). Both are light-sensitive channels that, whenexpressed and activated in neural tissue, allow for a cell to bedepolarized when stimulated with light (Boyden, 2005).

In some embodiments hybrid or chimeric channelrhodopsins can be createdand used by combining different portions of the ChR1 and ChR2 proteins.

In one embodiment a hybrid or chimeric channelrhodopsin can be createdand used by replacing the N-terminal segments of ChR2 with thehomologous counterparts of ChR1 (and vice-versa). In some embodimentsthe hybrid channelrhodopsins result in a shift of sensitivity into adifferent wavelength spectrum (for example into the red wavelengthspectrum) with negligible desensitization and slowed turning-on andturning-off kinetics.

In another embodiment a ChR1 (amino acids 1-345) and ChR2 (amino acids1-315) hybrid/chimera can be created and used.

In yet another embodiment ChR1-ChR2 hybrids/chimeras retaining theN-terminal portion of ChR1 and replacing the C-terminal portion with thecorresponding ChR2 segment can be created and used. In specificembodiments hybrids/chimeras of ChR1 and ChR2 can be constructed andutilized including mutant residues around the retinal binding pockets ofthe chimeras. In exemplary embodiments the following chimeras can becreated and used:

-   -   a. ChD: a hybrid/chimera of a ChR1 N-terminal portion and a ChR2        C-terminal portion where the crossover site is at a point of        homology at helixD of the two channelrhodopsins    -   b. ChEF: a hybrid/chimera of a ChR1 N-terminal portion and a        ChR2 C-terminal portion where the crossover site is at the loop        between helices E and F of the two channelrhodopsins    -   c. ChIEF: a variant of the ChEF chimera with isoleucine 170        mutated to valine    -   d. ChF: a hybrid/chimera of a ChR1 N-terminal portion and a ChR2        C-terminal portion where the crossover site is at the end of        helix F of the two channelrhodopsins.

In some embodiments the chimeras retain the reduced inactivation of ChR1in the presence of persistent light, but can allow the permeation ofsodium and potassium ions in addition to protons. In other embodimentsthe chimeras can improve the kinetics of the channel by enhancing therate of the channel closure after stimulation.

In some embodiments other ChR1 and ChR2 variants can be engineered. Inspecific embodiments single or multiple point mutations to the ChR2protein can result in ChR2 variants. In exemplary embodiments, mutationsat the C128 location of ChR2 can result in altered channel properties.In related embodiments, C128A, C128S, and C128T ChR2 mutations canresult in greater overall mean open times (Berndt, 2009). In otherrelated embodiments, ChR2 variants can result in altered kinetics.

In another embodiment, a VChR1 can be used (GenBank accession numberEU622855).

In specific embodiments a mammalian codon optimized version of ChR2 isutilized (FIG. 5).

NpHR (Halorhodopsin) (GenBank accession number EF474018; FIG. 3) is fromthe haloalkaliphilic archaeon Natronomonas pharaonis. In certainembodiments variants of NpHR can be created. In specific embodimentssingle or multiple point mutations to the NpHR protein can result inNpHR variants. In specific embodiments a mammalian codon optimizedversion of NpHR can be utilized.

In one embodiment NpHR variants are utilized. In one specific embodimenteNpHR (enhanced NpHR) is utilized. Addition of the amino acids FCYENEVto the NpHR C-terminus along with the signal peptide from the β subunitof the nicotinic acetylcholine receptor to the NpHR N-terminus resultsin the construction of eNpHR.

Melanopsin (GenBank accession number 6693702; FIG. 4) is a photopigmentfound in specialized photosensitive ganglion cells of the retina thatare involved in the regulation of circadian rhythms, pupillary lightreflex, and other non-visual responses to light. In structure,melanopsin is an opsin, a retinylidene protein variety ofG-protein-coupled receptor. Melanopsin resembles invertebrate opsins inmany respects, including its amino acid sequence and downstreamsignaling cascade. Like invertebrate opsins, melanopsin appears to be abistable photopigment, with intrinsic photoisomerase activity. Incertain embodiments variants of melanopsin can be created. In specificembodiments single or multiple point mutations to the melanopsin proteincan result in melanopsin variants.

Light-sensitive proteins may also include proteins that are at leastabout 10%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, or at leastabout 99% identical to the light-sensitive proteins ChR1, ChR2, NpHR andmelanopsin. For example, the ChR2 protein may include proteins that areat least about 60%, at least about 70%, at least about 80%, at leastabout 90% or at least about 95% identical to ChR2. In addition, theseproteins may include ChR2 that is photosensitive and can be activated byspecific wavelengths of high intensity light.

In some embodiments, light-sensitive proteins can modulate signalingwithin neural circuits and bidirectionally control behavior of ionicconductance at the level of a single neuron. In some embodiments theneuron is a retinal neuron, a retinal bipolar cell (e.g. ON or OFFretinal bipolar cells; rod and cone bipolar cells), a retinal ganglioncell, a photoreceptor cell, or a retinal amacrine cell.

Adeno-Associated Viral Vectors

The present invention provides viral vectors comprising nucleic acidsencoding a light-sensitive proteins and methods of use, as describedherein.

Adeno-associated virus (AAV) is a small (25-nm), nonenveloped virus thatpackages a linear single-stranded DNA genome of 4.7 Kb. The small sizeof the AAV genome and concerns about potential effects of Rep on theexpression of cellular genes led to the construction of AAV vectors thatdo not encode Rep and that lack the cis-active IEE, which is requiredfor frequent site-specific integration. The ITRs are kept because theyare the cis signals required for packaging. Thus, current recombinantAAV (rAAV) vectors persist primarily as extrachromosomal elements.

A variety of recombinant adeno-associated viral vectors (rAAV) may beused to deliver genes of interest to a cell and to effect the expressionof a gene of interest, e.g., a gene encoding a light-sensitive protein.For example, rAAV can be used to express light-sensitive proteins, e.g.,ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, andvariants thereof or any light-sensitive protein described herein, in atarget cell. At times herein, “transgene” is used to refer to apolynucleotide encoding a polypeptide of interest, wherein thepolynucleotide is encapsidated in a viral vector (e.g., rAAV).

Adeno-associated viruses are small, single-stranded DNA viruses whichrequire helper virus to facilitate efficient replication. The 4.7 kbgenome of AAV is characterized by two inverted terminal repeats (ITR)and two open reading frames which encode the Rep proteins and Capproteins, respectively. The Rep reading frame encodes four proteins ofmolecular weight 78 kD, 68 kD, 52 kD and 40 kD. These proteins functionmainly in regulating AAV replication and rescue and integration of theAAV into a host cell's chromosomes. The Cap reading frame encodes threestructural proteins of molecular weight 85 kD (VP 1), 72 kD (VP2) and 61kD (VP3) (Berns, cited above) which form the virion capsid. More than80% of total proteins in AAV virion comprise VP3.

The genome of rAAV is generally comprised of: (1) a 5′adeno-associatedvirus ITR, (2) a coding sequence (e.g., transgene) for the desired geneproduct (e. g., a light-sensitive protein) operatively linked to asequence which regulates its expression in a cell (e. g., a promotersequence such as a mGluR6 or fragment thereof), and (3) a3′adeno-associated virus inverted terminal repeat. In addition, the rAAVvector may preferably contain a polyadenylation sequence.

Generally, rAAV vectors have one copy of the AAV ITR at each end of thetransgene or gene of interest, in order to allow replication, packaging,and efficient integration into cell chromosomes. The ITR consists ofnucleotides 1 to 145 at the 5′end of the AAV DNA genome, and nucleotides4681 to 4536 (i. e., the same sequence) at the 3′end of the AAV DNAgenome. The rAAV vector may also include at least 10 nucleotidesfollowing the end of the ITR (i. e., a portion of the “D region”).

The transgene sequence (e.g., the polynucleotide encoding alight-sensitive protein) can be of about 2 to 5 kb in length (oralternatively, the transgene may additionally contain a “stuffer” or“filler” sequence to bring the total size of the nucleic acid sequencebetween the two ITRs to between 2 and 5 kb). Alternatively, thetransgene may be composed of repeated copies of the same or similarheterologous sequence several times (e. g., two nucleic acid moleculeswhich encode one or more light-sensitive proteins separated by aribosome readthrough, or alternatively, by an Internal Ribosome EntrySite or “IRES”), or several different heterologous sequences (e. g.,ChR2 and NpHR separated by a ribosome readthrough or an IRES; or any twoore more of the light-sensitive proteins described herein including butnot limited to ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T,ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR,melanopsin, and variants thereof).

Recombinant AAV vectors of the present invention may be generated from avariety of adeno-associated viruses, including for example, any ofserotypes 1 through 12, as described herein. For example, ITRs from anyAAV serotype are expected to have similar structures and functions withregard to replication, integration, excision and transcriptionalmechanisms.

In some embodiments, a cell-type specific promoter (or other regulatorysequence such as an enhancer) is employed to drive expression of a geneof interest, e.g., a light-sensitive protein, ChR2, etc., in one or morespecific cell types. In other cases, Within certain embodiments of theinvention, expression of the light-sensitive transgene may beaccomplished by a separate promoter (e. g., a viral, eukaryotic, orother promoter that facilitates expression of an operatively linkedsequence in an eukaryotic cell, particularly a mammalian cell).Representative examples of suitable promoters in this regard include amGluR6 promoter, a GNA01 promoter, a CBA/smCBA (fusion of the CMVimmediate early enhancer and bovine beta actin promoter plusintro1-exon1 junction) promoter, CBA promoter (chicken beta actin), CMVpromoter, RSV promoter, SV40 promoter, MoMLV promoter, or derivatives,mutants and/or fragments thereof. Promoters and other regulatorysequences are further described herein.

Other promoters that may similarly be utilized within the context of thepresent invention include cell or tissue specific promoters (e. g, arod, cone, or ganglia derived promoter), or inducible promoters.Representative examples of suitable inducible promoters includeinducible promoters sensitive to an antibiotic, e.g.,tetracycline-responsive promoters such as “tet-on” and/or “tet-off”promoters. Inducible promoters may also include promoters sensitive tochemicals other than antibiotics.

The rAAV vector may also contain additional sequences, for example froman adenovirus, which assist in effecting a desired function for thevector. Such sequences include, for example, those which assist inpackaging the rAAV vector into virus particles.

Packaging cell lines suitable for producing adeno-associated viralvectors may be accomplished given available techniques (see e. g., U.S.Pat. No. 5,872,005). Methods for constructing and packaging rAA7Ivectors are described in, for example, WO 00/54813.

Flanking the rep and cap open reading frames at the 5′ and 3′ ends are145 bp inverted terminal repeats (ITRs), the first 125 bp of which arecapable of forming Y- or T-shaped duplex structures. The two ITRs arethe only cis elements essential for AAV replication, rescue, packagingand integration of the AAV genome. There are two conformations of AAVITRs called “flip” and “flop”. These differences in conformationoriginated from the replication model of adeno-associated virus whichuses the ITR to initiate and reinitiate the replication (R. O. Snyder etal, 1993, J. Virol., 67:6096-6104 (1993); K. I. Berns, 1990Microbiological Reviews, 54:316-329). The entire rep and cap domains canbe excised and replaced with a therapeutic or reporter transgene.

In some embodiments self-complementary AAV vectors are used.Self-complementary vectors have been developed to circumventrate-limiting second-strand synthesis in single-stranded AAV vectorgenomes and to facilitate robust transgene expression at a minimal dose.In specific embodiments a self-complementary AAV of any serotype orhybrid serotype or mutant serotype, or mutant hybrid serotype increasesexpression of a light-sensitive protein such as ChR1, ChR2, VChR1, ChR2C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF,ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants thereof by at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 100%, at least 125%, at least150%, at least 175%, at least 200%, or more than 200%, when compared toa non-self complementary rAAV of the same serotype.

Adeno-Associated Viral Serotypes

In one embodiment the vector comprises a recombinant AAV of a particularserotype, either naturally occurring or engineered.

AAVs have been found in many animal species, including primates, canine,fowl and human.

Viral serotypes are strains of microorganisms having a set ofrecognizable antigens in common. There are several known serotypes ofAAV, and the efficacy of transfection or transduction within the retinamay vary as a function of the specific serotype and the nature of thetarget cells. rAAV, or a specific serotype of rAAV or AAV, may providetissue-specific or cell-type specific tropism for gene delivery toretinal bipolar cells (e.g. ON or OFF retinal bipolar cells; rod andcone bipolar cells). While rAAV and/or AAV is likely a relatively safemethod to deliver a transgene to a target tissue, the efficacy ofdelivery, and possibly the safety of delivery, may depend on the coatproteins of the AAV. The protein coat, or capsid, determines which cellscan take up the viral payload. Different AAV serotypes, i.e., virusesthat differ in their proteins coats or capsids, may differ in theirtissue tropism and ability to transduce targeted cells. Transgenes canbe packaged within AAV particles with many functionally different coatproteins, or capsids. These different capsids are what define theserotype and may contribute (entirely or in part) to its ability totransduce particular cell types. The entry of the viral vector beginswith the interaction of the capsid and the target cell surface proteins.Without wishing to be bound by theory, it is at this point in thetransduction pathway that different serotypes may significantlyinfluence the efficiency of transgene delivery.

In certain embodiments the AAV vector is of a serotype or variant/mutantthereof including but not limited to: AAV1 (GenBank accession numberAY724675), AAV2 (GenBank accession number AF043303), AAV3, AAV4, AAV5(GenBank accession number M61166), AAV6, AAV7 (GenBank accession numberAF513851), AAV8 (GenBank accession number AF513852), AAV9 (GenBankaccession number AX753250), AAV10, AAV11 (GenBank accession numberAY631966), or AAV12 (GenBank accession number DQ813647), or mutants,hybrids, or fragments thereof. In certain embodiments the AAV vectorcomprises one or more, two or more, three or more, four or more, or fiveor more of the following serotypes: AAV1 (GenBank accession numberAY724675), AAV2 (GenBank accession number AF043303), AAV3, AAV4, AAV5(GenBank accession number M61166), AAV6, AAV7 (GenBank accession numberAF513851), AAV8 (GenBank accession number AF513852), AAV9 (GenBankaccession number AX753250), AAV10, AAV11 (GenBank accession numberAY631966), or AAV12 (GenBank accession number DQ813647), or mutants,hybrids, or fragments thereof. In other embodiments, the AAV vector isof a natural serotype or variant/mutant thereof, heretofore yetundiscovered and uncharacterized.

In certain embodiments, the recombinant AAV is of a combinatorial hybridof 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more serotypes ormutants thereof.

In some embodiments, the AAV vector may be used to specificallytransduce a specific cell type, e.g., retinal cells or retinal bipolarcells (e.g. ON or OFF retinal bipolar cells; rod and cone bipolarcells). In some cases, a specific serotype, e.g., AAV2, AAV5, AAV7 orAAV8, may be better than other serotypes at transducing a particularcell type (e.g., retinal bipolar cells (e.g. ON or OFF retinal bipolarcells; rod and cone bipolar cells), neurons) or tissue. For example, aspecific AAV serotype such as AAV2, AAV5, AAV7 or AAV8 may transduce aspecific cell type, e.g., retinal bipolar cells (e.g. ON or OFF retinalbipolar cells; rod and cone bipolar cells), with an increasedtransduction efficiency of at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 100%, at least 125%, at least 150%, at least 175%, at least 200%,or more than 200%, when compared to a different AAV serotype. In somecases, a specific serotype e.g., AAV2, AAV5, AAV7 or AAV8, may permittransduction of at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,or 90% of cells of a particular cell type, e.g., retinal bipolar cells(e.g. ON or OFF retinal bipolar cells; rod and cone bipolar cells), orof cells within a particular tissue, e.g., retinal tissue.

There is a need in the art for AAV serotypes that can effectivelytransduce retinal bipolar cells (e.g. ON or OFF retinal bipolar cells;rod and cone bipolar cells), particularly when such transduction enablesthe delivery and expression of a light-sensitive protein, e.g., ChR2. Animportant therapy to treat eye disorders or diseases (e.g., visualimpairment, blindness), may involve using a particular AAV serotype toexpress light-sensitive proteins such as ChR2 in retinal bipolar cells(e.g. ON or OFF retinal bipolar cells; rod and cone bipolar cells). Forexample, in a preferred embodiment, AAV5 or AAV7 serotypes are used totarget expression of a gene of interest (e.g., a light-sensitiveprotein, ChR2, etc.) in retinal cells, e.g., retinal bipolar cells (e.g.ON or OFF retinal bipolar cells; rod and cone bipolar cells). In somecases, AAV5 and/or AAV7 may more efficiently transduce retinal cells,e.g., retinal bipolar cells (e.g. ON or OFF retinal bipolar cells; rodand cone bipolar cells), than other AAV serotypes. For example, in someembodiments, the AAV5 and/or AAV7 serotypes, but not AAV1 serotype, areused to transduce retinal bipolar cells (e.g. ON or OFF retinal bipolarcells; rod and cone bipolar cells). In other cases AAV2 and/or AAV8serotypes are used to transduce retinal bipolar cells (e.g. ON or OFFretinal bipolar cells; rod and cone bipolar cells). In some cases, aspecific serotype, e.g., AAV2, AAV5, AAV7 or AAV8, may be generallyapplied to a tissue, e.g., retinal tissue, but then preferentiallytransduces a specific cell-type over another cell type.

In some cases, an AAV e.g., AAV2, AAV5, AAV7 or AAV8, that is introducedto the retina may preferentially transduce retinal bipolar cells (e.g.ON or OFF retinal bipolar cells; rod and cone bipolar cells) so that thetransgene is expressed more highly in retinal bipolar cells compared toother retinal cells. In some cases, the particular serotype e.g., AAV2,AAV5, AAV7 or AAV8, of the AAV may be the cause or contribute to thecause of such preferential transduction. In some cases, only a smallsubset of the bipolar cells are transduced.

In some embodiments, a specific serotype of AAV, e.g., AAV5 and/or AAV7(or any other AAV serotype or mutant described herein) comprising anon-cell-type-specific promoter is used to drive expression of alight-sensitive protein in a particular cell type. In some cases, aspecific serotype of AAV that has been demonstrated to preferentiallytransduce a particular cell type is used along with a cell-type specificpromoter to drive expression of a protein of interest, e.g., alight-sensitive protein, in a specific cell-type.

The AAV ITR sequences and other AAV sequences employed in generating theminigenes, vectors, and capsids, and other constructs used in certainembodiments may be obtained from a variety of sources. For example, thesequences may be provided by presently identified human AAV types andAAV serotypes yet to be identified. Similarly, AAVs known to infectother animals may also provide these ITRs employed in the molecules orconstructs of this invention. Similarly, the capsids from a variety ofserotypes of AAV may be “mixed and matched” with the other vectorcomponents. See, e.g., International Patent Publication No. WO01/83692,published Nov. 8, 2001, and incorporated herein by reference. A varietyof these viral serotypes and strains are available from the AmericanType Culture Collection, Manassas, Va., or are available from a varietyof academic or commercial sources. Alternatively, it may be desirable tosynthesize sequences used in preparing the vectors and viruses of theinvention using known techniques, which may utilize AAV sequences whichare published and/or available from a variety of databases.

Adeno-Associated Viruses and Mutations of Surface-Exposed Residues

Recombinant adeno-associated virus vectors are in use in severalclinical trials, but relatively large vector doses are needed to achievetherapeutic benefits. Large vector doses may also trigger an immuneresponse as a significant fraction of the vectors may fail to trafficefficiently to the nucleus and may be targeted for degradation by thehost cell proteasome machinery. It has been reported that epidermalgrowth factor receptor protein tyrosine kinase (EGFR-PTK) signalingnegatively affects transduction by AAV Serotype 2 vectors by impairingnuclear transport of the vectors (Zhong 2007). Tyrosine-phosphorylatedAAV2 vectors enter but do not transduce effectively, in part because ofthe ubiquitination of AAV capsids followed by proteasome-mediateddegradation. Point mutations in tyrosines in AAV2 may lead tohigh-efficiency transduction at lower virus titers (Zhong 2008). In oneembodiment, tyrosine-mutated AAVs e.g., AAV2 or AAV8, are used in orderto improve the efficiency of transduction of retinal cells, e.g.,retinal bipolar cells (FIGS. 9-12). In one embodiment, mutations of thesurface-exposed tyrosine residues of rAAV capsid allow the vectors toevade phosphorylation and subsequent ubiquitination and, thus, preventproteasome-mediated degradation, leading to greater transduction andsubsequent gene expression of light-sensitive proteins.

In a related embodiment any one or more surface exposed residues otherthan tyrosine may be mutated to improve the transduction efficiency,tissue/cell-type tropism, expression characteristics, and titers neededfor effective infection.

As described herein, modification and changes to the structure of thepolynucleotides and polypeptides of wild-type rAAV vectors may result inimproved rAAV virions possessing desirable characteristics. For example,mutated rAAV vectors may improve delivery of light-sensitive geneconstructs to selected mammalian cell, tissues, and organs for thetreatment, prevention, and prophylaxis of various diseases anddisorders. Such approach may also provide a means for the ameliorationof symptoms of such diseases, and to facilitate the expression ofexogenous therapeutic and/or prophylactic polypeptides of interest viarAAV vector-mediated gene therapy. The mutated rAAV vectors may encodeone or more proteins, e.g., the light-sensitive proteins, e.g., ChR2,described herein. The creation (or insertion) of one or more mutationsinto specific polynucleotide sequences that encode one or more of thelight-sensitive proteins encoded by the disclosed rAAV constructs areprovided herein. In certain circumstances, the resulting light-sensitivepolypeptide sequence is altered by these mutations, or in other cases,the sequence of the polypeptide is unchanged by one or more mutations inthe encoding polynucleotide to produce modified vectors with improvedproperties for effecting gene therapy in mammalian systems. As describedherein, codon-optimization of the polynucleotide encoding thelight-sensitive protein may also improve transduction efficiency.

The ubiquitin-proteasome pathway plays a role in AAV-intracellulartrafficking. Substitution of surface exposed tyrosine residues on, forexample, AAV2 or AAV8 capsids permits the vectors to either have limitedubiquitination or to escape ubiquitination altogether. The reduction in,or absence of, ubiquitination may help prevent the capsid fromundergoing proteasome-mediated degradation. AAV or rAAV capsids can bephosphorylated at tyrosine residues by EGFR-PTK in an in vitrophosphorylation assay, and the phosphorylated AAV capsids retain theirstructural integrity. Although phosphorylated AAV vectors may entercells as efficiently as their unphosphorylated counterparts, theirtransduction efficiency may be significantly impaired.

In some cases, a recombinant adeno-associated viral (rAAV) vectorcomprises a capsid protein with a mutated tyrosine residue which enablesto the vector to have improved transduction efficiency of a target cell,e.g., a retinal bipolar cell (e.g. ON or OFF retinal bipolar cells; rodand cone bipolar cells). In some cases, the rAAV further comprises apromoter (e.g., mGluR6, or fragment thereof) capable of driving theexpression of a protein of interest in the target cell.

In some cases, expression in a specific cell type is further achieved byincluding a cell-type specific promoter described herein within the rAAVvector.

In one embodiment, a recombinant adeno-associated viral (rAAV) vectorcomprises at least a first capsid protein comprising at least a firstphosphorylated tyrosine amino acid residue, and wherein said vectorfurther comprises at least a first nucleic acid segment that encodes alight-sensitive protein operably linked to a promoter capable ofexpressing said segment in a host cell.

In one embodiment, a mutation may be made in any one or more of tyrosineresidues of the capsid protein of AAV 1-12 or hybrid AAVs. In specificembodiments these are surface exposed tyrosine residues. In a relatedembodiment the tyrosine residues are part of the VP1, VP2, or VP3 capsidprotein. In exemplary embodiments, the mutation may be made at one ormore of the following amino acid residues of an AAV-VP3 capsid protein:Tyr252, Tyr272, Tyr444, Tyr500, Tyr700, Tyr704, Tyr730; Tyr275, Tyr281,Tyr508, Tyr576, Tyr612, Tyr673 or Tyr720. Exemplary mutations aretyrosine-to-phenylalanine mutations including, but not limited to,Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F,Y576F, Y612G, Y673F and Y720F. In a specific embodiment these mutationsare made in the AAV2 serotype. In some cases, an AAV2 serotype comprisesa Y444F mutation and/or an AAV8 serotype comprises a Y733F mutation,wherein 444 and 733 indicate the location of a point tyrosine mutationof the viral capsid. In further embodiments, such mutated AAV2 and AAV8serotypes encode a light-sensitive protein, e.g., ChR2, and may alsocomprise a regulatory sequence (e.g., mGluR6) to drive expression ofsuch light-sensitive protein.

In a related embodiment, 1, 2, 3, 4, 5, 6, or 7 mutations are made tothe tyrosine residue on an AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, orhybrid serotype. In one exemplary embodiment, 3 tyrosines are mutated tocreate an AAV serotype with a triple mutation consisting of: Y444F,Y500F, and Y730F.

The rAAV vectors of the present invention may be comprised within anadeno-associated viral particle or infectious rAAV virion, including forexample, virions selected from the group consisting of an AAV serotype1, an AAV serotype 2, an AAV serotype 3, an AAV serotype 4, an AAVserotype 5 and an AAV serotype 6, an AAV serotype 7, an AAV serotype 8,an AAV serotype 9, an AAV serotype 10, an AAV serotype 11, an AAVserotype 12, or a hybrid AAV serotype.

The rAAV vectors of the present invention may also be comprised withinan isolated mammalian host cell, including for example, human, primate,murine, feline, canine, porcine, ovine, bovine, equine, epine, caprineand lupine host cells. The rAAV vectors may be comprised within anisolated mammalian host cell such as a human endothelial, epithelial,vascular, liver, lung, heart, pancreas, intestinal, kidney, muscle,bone, neural, blood, or brain cell.

In certain embodiments the transduction efficiency of an AAV comprisinga mutated capsid protein (e.g., a mutation of a tyrosine residuedescribed herein) expressing a light-sensitive protein such as ChR1,ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, andvariants thereof is increased by at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 100%, at least 125%, at least 150%, at least 175%, atleast 200%, or more than 200%, when compared to a wild-type AAVexpressing a light-sensitive protein. This disclosure also providesmutated rAAV vectors (e.g., the AAV2 Y444F vector or the AAV8Y733Fvector) capable of transducing at least 2%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90% of (e.g. ON or OFF retinal bipolar cells; rod andcone bipolar cells) bipolar cells. The improvement in transductioncreated by the mutated capsid may permit transduction of bipolar cellsby intravitreal injection. For example, in some embodiments, a mutatedrAAV vector or a rAAV combinatorial serotype hybrid vector or a mutatedcombinatorial serotype hybrid rAAV vector may be capable of transducingat least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% ofretinal bipolar cells (e.g. ON or OFF retinal bipolar cells; rod andcone bipolar cells) is introduced to the retina by intravitrealinjection. In a specific embodiment, only a subset of the retinalbipolar cells is transduced. In another specific embodiment only thehighly sensitive bipolar cells are transduced.

In certain embodiments the ubiquitin or proteasome-mediated degradationof an AAV comprising a capsid protein with a mutation expressing alight-sensitive protein, such as ChR1, ChR2, VChR1, ChR2 C128A, ChR2C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF,NpHR, eNpHR, melanopsin, and variants thereof, is decreased by at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, or at least 90%, when compared to a wild-type AAV expressing alight-sensitive protein.

Other Gene Delivery Vectors

Any of a variety of other vectors adapted for expression of ChR1, ChR2,VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras,ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants thereof orany light-sensitive protein in a cell of the eye, particularly within aretinal cell, more particularly within a non-photoreceptor cell (e.g.amacrine cells, retinal ganglion cells, retinal bipolar cells, (ON orOFF retinal bipolar cells; rod and cone bipolar cells)), are within thescope of the present invention. Gene delivery vectors can be viral (e.g., derived from or containing sequences of viral DNA or RNA, preferablypackaged within a viral particle), or non-viral (e. g., not packagedwithin a viral particle, including “naked” polynucleotides, nucleic acidassociated with a carrier particle such as a liposome or targetingmolecule, and the like).

Other exemplary gene delivery vectors are described below.

Recombinant Adenoviral Vectors (Ad):

in other embodiments, the gene delivery vector is a recombinantadenoviral vector. U.S. Pat. No. 6,245,330 describes recombinantadenoviruses which may be suitable for use in the invention. Ad vectorsdo not integrate into the host cell genome, particularly preferred whenshort term gene is required, typically about 14 days. Thus, use of Advectors can require repeated intraocular injections to treat a retinaldisease which continues over decades in the average patient.

The viral tropism of Ad and AAV in the retina is can be different. Thesubset of cells that are transduced by the vector is usually areceptor-mediated event. Ad vectors have been shown to primarilytransduce retinal Muller cells and Retinal pigment epithelial cellsfollowing injection. AAV vectors are very efficient at transferringtheir genetic payload to retinal photoreceptor and non-photoreceptorcells when injected into the eye.

Retroviral Gene Delivery Vectors:

the gene delivery vectors of the invention can be a retroviral genedelivery vector adapted to express a selected gene (s) or sequence (s)of interest (e. g., ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR,melanopsin, and variants thereof). Retroviral gene delivery vectors ofthe present invention may be readily constructed from a wide variety ofretroviruses, including for example, B, C, and D type retroviruses aswell as spumaviruses and lentiviruses. For example, in some cases, aretrovirus, e.g., a lentivirus, is pseudotyped with an envelope proteinor other viral protein to facilitate entry into target cells. In somecases, a lentivirus is pseudotyped with vesicular-stomatitis virus gprotein. (see RNA Tumor Viruses, Second Edition, Cold Spring HarborLaboratory, 1985). Such retroviruses may be readily obtained fromdepositories or collections such as the American Type Culture Collection(“ATCC”; Rockville, Md.), or isolated from known provided herein, andstandard recombinant techniques (e. g., Sambrook et al., MolecularCloning: A Laboratory Manual 2d ed., Cold Spring Harbor LaboratoryPress, 1989; Kunkel, PNAS 52: 488, 1985).

In addition, within certain embodiments of the invention, portions ofthe retroviral gene delivery vectors may be derived from differentretroviruses. For example, within one embodiment of the invention,retrovirus LTRs may be derived from a Murine Sarcoma Virus, a tRNAbinding site from a Rous Sarcoma Virus, a packaging signal from a MurineLeukemia Virus, and an origin of second strand synthesis from an AvianLeukosis Virus.

Within one aspect of the present invention, retroviral vector constructsare provided comprising a 5′LTR, a tRNA binding site, a packagingsignal, one or more heterologous sequences, an origin of second strandDNA synthesis and a 3′LTR, wherein the vector construct lacks gag, polor env coding sequences.

Other retroviral gene delivery vectors may likewise be utilized withinthe context of the present invention, and are well known in the art.

Packaging cell lines suitable for use with the above describedretroviral vector constructs can be readily prepared according tomethods well known in the art, and utilized to create producer celllines for the production of recombinant vector particles.

Alphavirus Delivery Vectors:

gene delivery vectors suitable for use in the invention can also bebased upon alphavirus vectors. For example, the Sindbis virus is theprototype member of the alphavirus genus of the togavirus family. Theunsegmented genomic RNA (49S RNA) of Sindbis virus is approximately11,703 nucleotides in length, contains a 5′cap and a 3′ poly-adenylatedtail, and displays positive polarity. Infectious enveloped Sindbis virusis produced by assembly of the viral nucleocapsid proteins onto theviral genomic RNA in the cytoplasm and budding through the cell membraneembedded with viral encoded glycoproteins. Entry of virus into cells isby endocytosis through clatharin coated pits, fusion of the viralmembrane with the endosome, release of the nucleocapsid, and uncoatingof the viral genome. During viral replication the genomic 49S RNA servesas template for synthesis of the complementary negative strand. Thisnegative strand in turn serves as template for genomic RNA and aninternally initiated 26S subgenomic RNA.

The Sindbis viral nonstructural proteins are translated from the genomicRNA while structural proteins are translated from the subgenomic 26SRNA. All viral genes are expressed as a polyprotein and processed intoindividual proteins by post translational proteolytic cleavage. Thepackaging sequence resides within the nonstructural coding region,therefore only the genomic 49S RNA is packaged into virions.

Several different Sindbis vector systems may be constructed and utilizedwithin the present invention. Representative examples of such systemsinclude those described within U.S. Pat. Nos. 5,091,309 and 5,217,879,and PCT Publication No. WO 95/07994.

Other viral gene delivery vectors In addition to retroviral vectors andalphavirus vectors, numerous other viral vectors systems may also beutilized as a gene delivery vector. Representative examples of such genedelivery vectors include viruses such as pox viruses, such as canary poxvirus or vaccinia virus

Non-Viral Gene Delivery Vectors:

in addition to the above viral-based vectors, numerous non-viral genedelivery vectors may likewise be utilized within the context of thepresent invention. Representative examples of such gene delivery vectorsinclude direct delivery of nucleic acid expression vectors, naked DNA(e. g., DNA not contained in a viral vector) (WO 90/11092), polycationcondensed DNA linked or unlined to killed adenovirus (Curiel et al.,Hum. Gene Ther. 3: 147-154, 1992), DNA ligand linked to a ligand with orwithout one of the high affinity pairs described above (Wu et al., R ofBiol. Chem (264: 16985-16987, 1989), nucleic acid containing liposomes(e. g., WO 95/24929 and WO 95/12387) and certain eukaryotic cells.

Regulatory Sequences

In some embodiments, regulatory sequences or elements are utilized toallow for cell-type or tissue-type specific targeting of ChR1, ChR2,VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras,ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants thereof. Inrelated embodiments regulatory elements are used to specifically targetretinal neurons, or retinal bipolar cells (e.g. ON or OFF retinalbipolar cells; rod and cone bipolar cells), or retinal ganglion cells,or photoreceptor cells, or amacrine cells. Examples of regulatorysequences or elements include, but are not limited to promoter,silencer, enhancer, and insulator sequences.

In some embodiments regulatory sequences such as promoters suitable foruse in the present invention include constitutive promoters, strongpromoters (e. g., CMV promoters), inducible promoters, andtissue-specific or cell-specific promoters (e. g., promoters thatpreferentially facilitate expression in a limited number of tissues orcell types (e. g., eye tissues, retina, retinal cells, photoreceptorcells, and the like).

Any of a variety of regulatory sequences can be used in the genedelivery vectors of the invention to provide for a suitable level orpattern of expression of the light-sensitive protein of interest. Theregulatory sequences are generally derived from eukaryotic regulatorysequences.

In some embodiments non-cell specific regulatory elements are used. Inone embodiment, the promoter comprises (from 5′ to 3′) a viral enhancer(a CMV immediate early enhancer), and a beta-actin promoter (a bovine orchicken beta-actin promoter-exon 1-intron 1 element). In a specificembodiment, the promoter comprises (from 5′ to 3′) CMV immediate earlyenhancer (381 bp)/bovine or chicken beta-actin (CBA) promoter-exon1-intron 1 (1352 bp) element, which together are termed herein the “CBApromoter” (FIG. 7). In some embodiments a nucleic acid encoding alight-sensitive protein is delivered to a cell using a viral vector suchas AAV carrying a selected light-sensitive transgene-encoding DNAregulated by a non cell-specific promoter and/or other regulatorysequences that expresses the product of the DNA. In some relatedembodiments the non cell-specific promoter is general promoter such as aubiquitin-based promoter, for e.g. a ubiquitin C promoter.

In other embodiments, a light-sensitive protein is delivered to a celltype or tissue type of interest using a viral vector such as AAVcarrying a selected light-sensitive transgene-encoding DNA regulated bya promoter and/or other regulatory sequences that expresses the productof the DNA in selected retinal cells of a subject. In specificembodiments, expression is targeted to particular types of cells withinthe retina through the use of a specific promoter nucleotide sequenceand/or other regulatory regions such as silencer, enhancer, or insulatorsequences which are engineered into the vector. In some embodiments,different regulatory sequences are used to drive expression of differentengineered genes in different populations of cells.

In other embodiments retinal bipolar cell-specific regulatory sequencessuch as promoter, enhancer, silencer, and insulator sequences are used.In specific embodiments the ON bipolar cells are targeted. In otherembodiments the OFF bipolar cells are targeted. In other embodiments therode bipolar cells are targeted. In other embodiments the cone bipolarcells are targeted.

In one embodiment specific expression of a light sensitive proteins inON bipolar cells is targeted using a light-sensitive protein such asChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, andvariants thereof operatively linked to a GRM6 (metabotropic glutamatereceptor 6, mGluR6) regulatory sequence or a fragment thereof. In oneembodiment the full length mGluR6 regulatory sequence is utilized.

In another embodiment specific expression of a light sensitive proteinsin ON bipolar cells is targeted using a light-sensitive protein such asChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, andvariants thereof operatively linked to a mGluR6 regulatory sequencefragment. In a specific embodiment, the mGluR6 regulatory sequencefragment is the sequence presented in FIG. 6. In a related embodimentthe mGluR6 regulatory sequence fragment is substantially the same as thesequence presented in FIG. 6, or is about 60% identical, or is about 70%identical, or is about 80% identical, or is about 90% identical, or isabout 95% identical to the sequence presented in FIG. 6.

Many known promoters are too large to fit into the genome of the AAV.Indeed, the original cell-specific regulatory sequence for mGluR6(Dhingra, 2008) was far too large (approximately 10.5 Kb) to be used inAAV. In a preferred embodiment of the current invention, a mGluR6regulatory sequence fragment is used, one that is small enough to beused in AAV-mediated delivery. In related embodiments the mGluR6regulatory sequence fragment is less than about 2000 base pairs, lessthan about 1000 base pairs, less than about 750 base pairs, less thanabout 500 base pairs, less than about 250 base pairs, or less than about100 base pairs in length. In another related embodiment, the mGluR6regulatory sequence fragment is a variant of the sequence presented inFIG. 6.

In another embodiment, specific expression of a light sensitive proteinsin ON bipolar cells is targeted using a light-sensitive protein such asChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, andvariants thereof operatively linked to a GNA01 (guanine nucleotidebinding protein (G protein), alpha activating activity polypeptide O)regulatory sequence. In a related embodiment the regulatory sequence issubstantially the same as GNA01 sequence, or is about 60% identical, oris about 70% identical, or is about 80% identical, or is about 90%identical, or is about 95% identical to the GNA01 sequence.

Retinal Bipolar Cells and Targeting

Although only a fraction of the human visual system, the retina is acomplex system that filters, amplifies, and modulates the visual signalbefore it is sent to the rest of the visual system (Wassle 2004). Thevast majority of this processing happens within the inner plexiformlayer (IPL) where a system of bipolar and amacrine cells refine thevisual signal into its primary components (e.g., motion, contrast,resolution) (Mills 1999; Roska 2001). Some groups are currentlytargeting ChR2 to the ganglion cell layer, which bypasses the processingpower of the IPL and system of amacrine cells (Bi 2006; Greenberg 2007;Tomita 2007). These groups have reported very few behavioral changes.

The majority of retinal cells are either ON-center (increased firingrate as a result of a step increase in contrast within the center of thereceptive field) or OFF center type (increased firing rate as a resultof a step decrease in contrast in the center of the receptive field),working in a push-pull inhibitory fashion (Wassle 2004). In order tomaintain this relationship between the two pathways, these two pathwayscan be driven independently. ON and OFF channels of informationtraveling from bipolar to ganglion cells are partially modulated througha network of inhibitory amacrine cells within the inner nuclear layer(Roska 2001). This bipolar-amacrine network produces temporally-distinctparallel channels of information: sustained-activity neurons, forexample, maintain activity throughout the light step, whereastransient-activity neurons have activity only at the onset or offset.These distinct patterns of response code for visual informationluminance, shape, edges, and motion (Wassle 2004). In some embodiments,cells that are pre-synaptic to the retinal ganglion cells aregenetically targeted to maintain the naturalism of these pathways andelicit naturalistic ganglion cell spiking.

In some embodiments, retinal bipolar cells (e.g. ON or OFF retinalbipolar cells; rod and cone bipolar cells) are genetically targeted.Targeting retinal bipolar cells may allow the retina to respond toexternal light and, more importantly, convey meaningful imageinformation to the brain even in the absence of natural photoreceptors.

Conditions Amenable to Treatment

In some embodiments, the present invention provides methods of treatinga subject suffering from a disease or disorder. The compositions andmethods described herein can be utilized to treat central and peripheralnervous system diseases and disorders.

In one aspect, the compositions and methods of this invention areutilized to treat photoreceptor diseases. Photoreceptor diseases such asretinitis pigmentosa (RP) and age-related macular degeneration (ARMD)cause blindness (Congdon 2004) in 15 million people worldwide (Chader2002), a number that is increasing with the age of the population. Therehave been attempts to restore basic visual function through genereplacement therapy or cellular transplantation (Acland 2001, Acland2005, Batten 2005, Pawlyk 2005, Aguirre 2007, MacLaren 2006). However,current approaches are fundamentally limited in scope and extent ofpotential impact, as they attempt to correct mechanistically distinctgenetic pathways on a one-at-a-time basis (Punzo 2007). Photoreceptordiseases are genetically diverse, with over 160 different mutationsleading to degeneration (Punzo 2007). There have also been efforts inutilizing electrical stimulation with implanted acute, semi-acute, andlong-term retinal prostheses in human subjects (de Balthasar, 2008;Horsager, 2009). They have shown elementary progress but aregene-nonspecific; electrical stimulation offers only gross specificityand indiscriminately drives visual information channels mediated byunique cell types. Activating retinal neurons requires large discelectrodes (at least 20 times the diameter of a retinal ganglion cell),leading to stimulation of broad areas of retina in a nonselectivefashion, greatly limiting the achievable visual resolution (Winter2007). In this aspect, the compositions and methods of this inventionconsist of introducing a gene encoding a light-sensitive protein (e.g.,ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, andvariants thereof) to induce light sensitivity in 2^(nd) order neurons(e.g., bipolar cells) delivered using a viral vector such as an AAV8with a single tyrosine to phenylalanine mutation, under the control of aregulatory element (e.g., GRM6). The activation of these light-sensitiveproteins could be controlled by ambient light or through alight-delivery device such as the goggles described in FIG. 13.

The methods of the invention can be used to treat (e. g., prior to orafter the onset of symptoms) in a susceptible subject or subjectdiagnosed with a variety of eye diseases. The eye disease may be aresults of environmental (e. g., chemical insult, thermal insult, andthe like), mechanical insult (e. g., injury due to accident or surgery),or genetic factors. The subject having the condition may have one orboth eyes affected, and therapy may be administered according to theinvention to the affected eye or to an eye at risk of photoreceptordegeneration due to the presence of such a condition in the subject'sother, affected eye.

The present invention provides methods which generally comprise the stepof intraocularly administering (e. g., by subretinal injection or byintravitreal injection) a gene delivery vector which directs theexpression of a light-sensitive protein to the eye to treat, prevent, orinhibit the onset or progression of an eye disease. As utilized herein,it should be understood that the terms “treated, prevented, or,inhibited” refers to the alteration of a disease onset, course, orprogress in a statistically significant manner.

Another condition amenable to treatment according to the invention isAge-related Macular Degeneration (AMD). The macula is a structure nearthe center of the retina that contains the fovea. This specializedportion of the retina is responsible for the high-resolution vision thatpermits activities such as reading. The loss of central vision in AMD isdevastating. Degenerative changes to the macula (maculopathy) can occurat almost any time in life but are much more prevalent with advancingage. Conventional treatments are short-lived, due to recurrent choroidalneovascularization. AMD has two primary pathologic processes, choroidalneovascularization (CNV) and macular photoreceptor cell death.

Exemplary conditions of particular interest which are amenable totreatment according to the methods of the invention include, but are notnecessarily limited to, retinitis pigmentosa (RP), diabetic retinopathy,and glaucoma, including open-angle glaucoma (e. g., primary open-angleglaucoma), angle-closure glaucoma, and secondary glaucomas (e. g.,pigmentary glaucoma, pseudoexfoliative glaucoma, and glaucomas resultingfrom trauma and inflammatory diseases).

Further exemplary conditions amenable to treatment according to theinvention include, but are not necessarily limited to, retinaldetachment, age-related or other maculopathies, photic retinopathies,surgery-induced retinopathies, toxic retinopathies, retinopathy ofprematurity, retinopathies due to trauma or penetrating lesions of theeye, inherited retinal degenerations, surgery-induced retinopathies,toxic retinopathies, retinopathies due to trauma or penetrating lesionsof the eye.

Specific exemplary inherited conditions of interest for treatmentaccording to the invention include, but are not necessarily limited to,Bardet-Biedl syndrome (autosomal recessive); Congenital amaurosis(autosomal recessive); Cone or cone-rod dystrophy (autosomal dominantand X-linked forms); Congenital stationary night blindness (autosomaldominant, autosomal recessive and X-linked forms); Macular degeneration(autosomal dominant and autosomal recessive forms); Optic atrophy,autosomal dominant and X-linked forms); Retinitis pigmentosa (autosomaldominant, autosomal recessive and X-linked forms); Syndromic or systemicretinopathy (autosomal dominant, autosomal recessive and X-linkedforms); and Usher syndrome (autosomal recessive).

In another aspect, the compositions and methods of this invention areutilized to treat peripheral injury, nociception, or chronic pain.Nociception (pain) for prolonged periods of time can give rise tochronic pain and may arise from injury or disease to visceral, somaticand neural structures in the body. Although the range of pharmacologicaltreatments for neuropathic pain has improved over the past decade, manypatients do not get effective analgesia, and even effective medicationsoften produce undesirable side effects. Substance P (SP) is involved innociception, transmitting information about tissue damage fromperipheral receptors to the central nervous system to be converted tothe sensation of pain. It has been theorized that it plays a part infibromyalgia A role of substance P in nociception is suggested by thereduction in response thresholds to noxious stimuli by centraladministration of NK1 and NK2 agonists. Pain behaviors induced bymechanical, thermal and chemical stimulation of somatic and visceraltissues were reduced in the mutant mice lacking SP/NKA. In oneembodiment light-sensitive proteins can silence the activity ofover-active neurons (i.e., substance P expressing peripheral neurons)due to peripheral injury or chronic pain using NpHR or eNpHR. NpHR/eNpHRcan be genetically targeted to substance P expressing cells using thesubstance P promoter sequence. In another embodiment light-sensitiveproteins enhance the activity of neurons that are inactive due toperipheral injury or chronic pain.

In another aspect, the compositions and methods of this invention areutilized to treat spinal cord injury and/or motor neuron diseases.Spinal cord injury can cause myelopathy or damage to white matter andmyelinated fiber tracts that carry sensation and motor signals to andfrom the brain. It can also damage gray matter in the central part ofthe spine, causing segmental losses of interneurons and motor neurons.Spinal cord injury can occur from many causes, including but not limitedto trauma, tumors, ischemia, abnormal development, neurodevelopmental,neurodegenerative disorders or vascular malformations. In one embodimentlight-sensitive proteins activate damaged neural circuits to restoremotor or sensory function. In one specific embodiment the elements actto allow control of autonomic and visceral functions. In otherembodiments the elements act to allow control of somatic skeletalfunction. The neural control of storage and voiding of urine is complexand dysfunction can be difficult to treat. One treatment for people withrefractory symptoms is continuous electrical nerve stimulation of thesacral nerve roots using implanted electrodes and an implanted pulsegenerator. However, stimulation of this nerve root can result in anumber of different complications or side effects. Being able todirectly control the sacral nerve through genetically-targeted toolswould be highly beneficial. In one embodiment, both ChR2 and NpHR couldbe expressed in this nerve to control storage and voiding of thebladder.

In another aspect, the compositions and methods of this invention areutilized to treat Parkinson's disease. Parkinson's disease belongs to agroup of conditions called movement disorders. They are characterized bymuscle rigidity, tremor, a slowing of physical movement (bradykinesia)and, in extreme cases, a loss of physical movement (akinesia). Theprimary symptoms are the results of decreased stimulation of the motorcortex by the basal ganglia, normally caused by the insufficientformation and action of dopamine, which is produced in the dopaminergicneurons of the brain. Parkinson's disease is both chronic andprogressive. Deep brain stimulation (DBS) is an effective surgicaltreatment for advanced Parkinson's disease (PD), with significantadvantages in morbidity-mortality and quality of life when compared tolesion techniques such as thalamotomy and/or pallidotomy. The procedureis indicated in patients with severe resting tremor, unresponsive toconventional medical treatment or with motor complications. The mostcommonly reported complications in the intra- and post-surgical periodare aborted procedure, misplaced leads, intracranial hemorrhage,seizures and hardware complications, whereas in the long-term period,symptoms may include high level cognitive dysfunction, psychiatric, andsubtle language problems. Indeed, this method of therapy would beimproved by being able to target specific cell types within a givenregion to avoid these side effects. In one embodiment light-sensitiveproteins specifically activate dopaminergic circuits.

In another specific aspect, the compositions and methods of thisinvention are utilized to treat epilepsy and seizures. Epilepsy is aneurological disorder that is often characterized by seizures. Theseseizures are transient signs and/or symptoms due to abnormal, excessiveor asynchronous neuronal activity in the brain. Over 30% of people withepilepsy do not have seizure control even with the best availablemedications. Epilepsy is not a single disorder, but rather as a group ofsyndromes with vastly divergent symptoms but all involving episodicabnormal electrical activity in the brain. Acute deep brain stimulation(DBS) in various thalamic nuclei and medial temporal lobe structures hasrecently been shown to be efficacious in small pilot studies. There islittle evidence-based information on rational targets and stimulationparameters. Amygdalohippocampal DBS has yielded a significant decreaseof seizure counts and interictal EEG abnormalities during long-termfollow-up. Data from pilot studies suggest that chronic DBS for epilepsymay be a feasible, effective, and safe procedure. Again, being able togenetically-target activation to specific subsets of cells would improvethe quality of the therapy as well as minimize overall side effects. Inspecific embodiments, the light-sensitive proteins are utilized to alterthe asynchronous electrical activity leading to seizures in these deepbrain areas.

In another aspect the compositions and methods of this invention areutilized to effect the light-stimulated release of implanted drug orvaccine stores for the prevention, treatment, and amelioration ofdiseases.

In another aspect the compositions and methods of this invention areutilized to treat neurodegenerative disease selected from but notlimited to alcoholism, Alexander's disease, Alper's disease, Alzheimer'sdisease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Battendisease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovinespongiform encephalopathy (BSE), chronic pain, Canavan disease, Cockaynesyndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease,Huntington's disease, HIV-associated dementia, Kennedy's disease,Krabbe's disease, Lewy body dementia, Machado-Joseph disease(Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple SystemAtrophy, Narcolepsy, Neuroborreliosis, Parkinson's disease,Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis,Prion diseases, Refsum's disease, Sandhoffs disease, Schilder's disease,Subacute combined degeneration of spinal cord secondary to PerniciousAnaemia, Schizophrenia, Spielmeyer-Vogt-Sjogren-Batten disease (alsoknown as Batten disease), Spinocerebellar ataxia (multiple types withvarying characteristics), Spinal muscular atrophy,Steele-Richardson-Olszewski disease, and Tables dorsalis.

In another aspect the compositions and methods of this invention areutilized to treat a neurodevelopmental disease selected from but notlimited to attention deficit hyperactivity disorder (ADHD), attentiondeficit disorder (ADD), schizophrenia, obsessive-compulsive disorder(OCD), mental retardation, autistic spectrum disorders (ASD), cerebralpalsy, Fragile-X Syndrome, Downs Syndrome, Rett's Syndrome, Asperger'ssyndrome, Williams-Beuren Syndrome, childhood disintegrative disorder,articulation disorder, learning disabilities (i.e., reading orarithmetic), dyslexia, expressive language disorder and mixedreceptive-expressive language disorder, verbal or performance aptitude.Diseases that can result from aberrant neurodevelopmental processes canalso include, but are not limited to bi-polar disorders, anorexia,general depression, seizures, obsessive compulsive disorder (OCD),anxiety, bruixism, Angleman's syndrome, aggression, explosive outburst,self injury, post traumatic stress, conduct disorders, Tourette'sdisorder, stereotypic movement disorder, mood disorder, sleep apnea,restless legs syndrome, dysomnias, paranoid personality disorder,schizoid personality disorder, schizotypal personality disorder,antisocial personality disorder, borderline personality disorder,histrionic personality disorder, narcissistic personality disorder,avoidant personality disorder, dependent personality disorder, reactiveattachment disorder; separation anxiety disorder; oppositional defiantdisorder; dyspareunia, pyromania, kleptomania, trichotillomania,gambling, pica, neurotic disorders, alcohol-related disorders,amphetamine-related disorders, cocaine-related disorders, marijuanaabuse, opioid-related disorders, phencyclidine abuse, tobacco usedisorder, bulimia nervosa, delusional disorder, sexual disorders,phobias, somatization disorder, enuresis, encopresis, disorder ofwritten expression, expressive language disorder, mental retardation,mathematics disorder, transient tic disorder, stuttering, selectivemutism, Crohn's disease, ulcerative colitis, bacterial overgrowthsyndrome, carbohydrate intolerance, celiac sprue, infection andinfestation, intestinal lymphangiectasia, short bowel syndrome, tropicalsprue, Whipple's disease, Alzheimer's disease, Parkinson's Disease, ALS,spinal muscular atrophies, and Huntington's Disease. Further examples,discussion, and information on neurodevelopmental disorders can befound, for example, through the Neurodevelopmental Disorders Branch ofthe National Institute of Mental Health (worldwide website address atnihm.nih.gov/dptr/b2-nd.cfm). Additional information onneurodevelopmental disorders can also be found, for example, inDevelopmental Disabilities in Infancy and Childhood: NeurodevelopmentalDiagnosis and Treatment, Capute and Accardo, eds. 1996, Paul H BrookesPub Co.; Hagerman, Neurodevelopmental Disorders: Diagnosis andTreatment, 1999, Oxford Univ Press; Handbook of Neurodevelopmental andGenetic Disorders in Children, Goldstein and Reynolds, eds., 1999,Guilford Press; Handbook of Neurodevelopmental and Genetic Disorders inAdults, Reynolds and Goldstein, eds., 2005, Guilford Press; andNeurodevelopmental Disorders, Tager-Flusberg, ed., 1999, MIT Press.

Assessment of Therapy

The effects of therapy according to the invention as described hereincan be assessed in a variety of ways, using methods known in the art.For example, the subject's vision can be tested according toconventional methods. Such conventional methods include, but are notnecessarily limited to, electroretinogram (ERG), focal ERG, tests forvisual fields, tests for visual acuity, ocular coherence tomography(OCT), Fundus photography, Visual Evoked Potentials (VEP) andPupillometry. In other embodiments, the subject can be assessedbehaviorally. In general, the invention provides for maintenance of asubject's vision (e. g., prevention or inhibition of vision loss offurther vision loss due to photoreceptor degeneration), slows onset orprogression of vision loss, or in some embodiments, provides forimproved vision relative to the subject's vision prior to therapy.

Methods of Administration

The gene delivery vectors of the present invention can be delivered tothe eye through a variety of routes. They may be deliveredintraocularly, by topical application to the eye or by intraocularinjection into, for example the vitreous (intravitreal injection) orsubretinal (subretinal injection) inter-photoreceptor space.Alternatively, they may be delivered locally by insertion or injectioninto the tissue surrounding the eye. They may be delivered systemicallythrough an oral route or by subcutaneous, intravenous or intramuscularinjection. Alternatively, they may be delivered by means of a catheteror by means of an implant, wherein such an implant is made of a porous,non-porous or gelatinous material, including membranes such as silasticmembranes or fibers, biodegradable polymers, or proteinaceous material.The gene delivery vector can be administered prior to the onset of thecondition, to prevent its occurrence, for example, during surgery on theeye, or immediately after the onset of the pathological condition orduring the occurrence of an acute or protracted condition.

In another embodiment the inner limiting membrane (ILM) is broken downto effect delivery. The ILM is the boundary between the retina and thevitreous body, formed by astrocytes and the end feet of Muller cells. Inboth nonhuman primates and humans, the ILM is thick and provides asubstantial barrier to the retina. Indeed, using intravitrealinjections, most viral particles are incapable of transducing retinalcells. In one embodiment, to improve transduction efficiency, an ILMpeel is conducted comprising carrying out a surgical procedure thatcomprises peeling off a small part of the ILM. In another embodiment, toimprove transduction efficiency, the ILM barrier can be partially orwholly broken down comprising using enzymatic techniques and one or moreenzymes.

In one embodiment the ILM is maintained to limit the therapeutic effectof the light-sensitive protein to the macula. In another embodiment theILM peel procedure and/or the ILM enzymatic digestion procedure, bothdescribed herein is used to achieve a broader distribution of thelight-sensitive protein.

The gene delivery vector can be modified to enhance penetration of theblood-retinal barrier. Such modifications may include increasing thelipophilicity of the pharmaceutical formulation in which the genedelivery vector is provided.

The gene delivery vector can be delivered alone or in combination, andmay be delivered along with a pharmaceutically acceptable vehicle.Ideally, such a vehicle would enhance the stability and/or deliveryproperties. The invention also provides for pharmaceutical compositionscontaining the active factor or fragment or derivative thereof, whichcan be administered using a suitable vehicle such as liposomes,microparticles or microcapsules. In various embodiments of theinvention, it may be useful to use such compositions to achievesustained release of the active component.

The amount of gene delivery vector (e. g., the number of viralparticles), and the amount of light-sensitive protein expressed,effective in the treatment of a particular disorder or condition maydepend of the nature of the disorder or condition and a variety ofpatient-specific factors, and can be determined by standard clinicaltechniques.

In one embodiment, the gene delivery vectors are administered to theeye, intraocularly to a variety of locations within the eye depending onthe type of disease to be treated, prevented, or, inhibited, and theextent of disease. Examples of suitable locations include the retina (e.g., for retinal diseases), the vitreous, or other locations in oradjacent the retina or in or adjacent the eye.

The human retina is organized in a fairly exact mosaic. In the fovea,the mosaic is a hexagonal packing of cones. Outside the fovea, the rodsbreak up the close hexagonal packing of the cones but still allow anorganized architecture with cones rather evenly spaced surrounded byrings of rods. Thus in terms of densities of the different photoreceptorpopulations in the human retina, it is clear that the cone density ishighest in the foveal pit and falls rapidly outside the fovea to afairly even density into the peripheral retina (see Osterberg, G. (1935)Topography of the layer of rods and cones in the human retina. ActaOphthal. (suppl.) 6, 1-103; see also Curcio, C. A., Sloan, K. R.,Packer, O., Hendrickson, A. E. and Kalina, R. E. (1987) Distribution ofcones in human and monkey retina: individual variability and radialasymmetry. Science 236, 579-582).

Access to desired portions of the retina, or to other parts of the eyemay be readily accomplished by one of skill in the art (see, generallyMedical and Surgical Retina: Advances, Controversies, and Management,Hilel Lewis, Stephen J. Ryan, Eds., medical-“illustrator, Timothy C.Hengst. St. Louis: Mosby, c1994. xix, 534; see also Retina, Stephen J.Ryan, editor in chief, 2nd ed., St. Louis, Mo.: Mosby, c1994. 3 v.(xxix, 2559).

In one embodiment, the amount of the specific viral vector applied tothe retina is uniformly quite small as the eye is a relatively containedstructure and the agent is injected directly into it. The amount ofvector that needs to be injected is determined by the intraocularlocation of the chosen cells targeted for treatment. The cell type to betransduced may be determined by the particular disease entity that is tobe treated.

For example, a single 20-microliter volume (e. g., containing about 10¹³physical particle titer/ml rAAV) may be used in a subretinal injectionto treat the macula and fovea of a human eye. A larger injection of 50to 100 microliters may be used to deliver the rAAV to a substantialfraction of the retinal area, perhaps to the entire retina dependingupon the extent of lateral spread of the particles.

A 100 microliter injection may provide several million active rAAVparticles into the subretinal space. This calculation is based upon atiter of 10¹³ physical particles per milliliter. Of this titer, it isestimated that 1/1000 to 1/10,000 of the AAV particles are infectious.The retinal anatomy constrains the injection volume possible in thesubretinal space (SRS). Assuming an injection maximum of 100microliters, this could provide an infectious titer of 10⁵ to 10⁹ rAAVin the SRS. This would have the potential of infecting all of theapproximately 150×10⁶ photoreceptors in the entire human retina with asingle injection.

Smaller injection volumes focally applied to the fovea or macula mayadequately transfect the entire region affected by the disease in thecase of macular degeneration or other regional retinopathies.

Depending on the application at least 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or more particles can be delivered intothe tissue of interest.

Gene delivery vectors can alternately be delivered to the eye byintraocular injection into the vitreous, e. g., to treat glaucomatousloss of retinal ganglion cells through apoptosis. In the treatment ofglaucoma, the primary target cells to be transduced are the retinalganglion cells, the retinal cells primarily affected. In such anembodiment, the injection volume of the gene delivery vector could besubstantially larger, as the volume is not constrained by the anatomy ofthe subretinal space. Acceptable dosages in this instance can range fromabout 25 microliters to 1000 microliters.

Pharmaceutical Compositions

Gene delivery vectors can be prepared as a pharmaceutically acceptablecomposition suitable for administration. In general, such pharmaceuticalcompositions comprise an amount of a gene delivery vector suitable fordelivery of light-sensitive protein-encoding polynucleotide to a cell ofthe eye for expression of a therapeutically effective amount of thelight-sensitive protein, combined with a pharmaceutically acceptablecarrier or excipient. Preferably, the pharmaceutically acceptablecarrier is suitable for intraocular administration. Exemplarypharmaceutically acceptable carriers include, but are not necessarilylimited to, saline or a buffered saline solution (e. g.,phosphate-buffered saline).

Various pharmaceutically acceptable excipients are well known in theart. As used herein, “pharmaceutically acceptable excipient” includesany material which, when combined with an active ingredient of acomposition, allows the ingredient to retain biological activity,preferably without causing disruptive reactions with the subject'simmune system or adversely affecting the tissues surrounding the site ofadministration (e. g., within the eye).

Exemplary pharmaceutically carriers include sterile aqueous ofnon-aqueous solutions, suspensions, and emulsions. Examples include, butare not limited to, any of the standard pharmaceutical excipients suchas a saline, buffered saline (e. g., phosphate buffered saline), water,emulsions such as oil/water emulsion, and various types of wettingagents.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, hyaluronic acid, vegetable oils such as olive oil, andinjectable organic esters such as ethyl oleate.

Aqueous carriers include water, alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles caninclude fluid and nutrient replenishers, electrolyte replenishers (suchas those based on Ringer's dextrose), and the like.

A composition of gene delivery vector of the invention may also belyophilized using means well known in the art, for subsequentreconstitution and use according to the invention. Where the vector isto be delivered without being encapsulated in a viral particle (e. g.,as “naked” polynucleotide), formulations for liposomal delivery, andformulations comprising microencapsulated polynucleotides, may also beof interest.

Compositions comprising excipients are formulated by well knownconventional methods (see, for example, Remington's PharmaceuticalSciences, Chapter 43, 14th Ed., Mack Publishing Col, Easton Pa. 18042,USA).

In general, the pharmaceutical compositions can be prepared in variousforms, preferably a form compatible with intraocular administration.Stabilizing agents, wetting and emulsifying agents, salts for varyingthe osmotic pressure or buffers for securing an adequate pH value mayalso optionally be present in the pharmaceutical composition.

The amount of gene delivery vector in the pharmaceutical formulationscan vary widely, i. e., from less than about 0.1%, usually at or atleast about 2% to as much as 20% to 50% or more by weight, and may beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected.

The pharmaceutical composition can comprise other agents suitable foradministration, which agents may have similar to additionalpharmacological activities to the light-sensitive protein to bedelivered (e. g., ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T,ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR,melanopsin, and variants thereof).

Kits

The invention also provides kits comprising various materials forcarrying out the methods of the invention. In one embodiment, the kitcomprises a vector encoding a light-sensitive protein polypeptide (e.g.ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, andvariants thereof), which vector is adapted for delivery to a subject,particularly an eye of the subject, and adapted to provide forexpression of the light-sensitive polypeptide in a cell of an eye,particularly a mammalian cell. The kit can comprise the vector in asterile vial, which may be labeled for use. The vector can be providedin a pharmaceutical composition. In one embodiment, the vector ispackaged in a virus. The kit can further comprise a needle and/orsyringe suitable for use with the vial or, alternatively, containing thevector, which needle and/or syringe are preferably sterile. In anotherembodiment, the kit comprises a catheter suitable for delivery of avector to the eye, which catheter may be optionally attached to asyringe for delivery of the vector. The kits can further compriseinstructions for use, e. g., instructions regarding route ofadministration, dose, dosage regimen, site of administration, and thelike.

Devices

The data in FIG. 12C demonstrate that the delivery of light-sensitiveproteins can work in the range of normal vision. In some embodiments,for greater efficacy, an internal or external device may be used. In oneembodiment an external device, such as a goggle, can be used forgeneration and/or amplification of light. In embodiments where a subjecthaving partial vision is being treated, i.e. a treatment of a subjectwhose photoreceptors are only partially damaged and a light-sensitiveprotein such as ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T,ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR,melanopsin, and variants thereof is being delivered, the stimulation maybe adjusted so that the surviving and/or healthy photoreceptors are notoverdriven by the light generation/amplification device. In variousembodiments, limiting overdriving by the light generation/amplificationdevice can be achieved by i) stimulating evenly, but shielding thesurviving or healthy photoreceptor cells from bright light through animplanted or external contact-lens type partial sunglass (tinting overphotoreceptors, clear over light-sensitive protein transduction area);ii) adjustment of the stimulation intensity to match the cell typesbeing stimulated; or iii) adjustment of the stimulation to the be nearthe top of the visual dynamic range.

In one embodiment, an internal light-generating device is implanted.

In another embodiment, a protective optic, or a contact lens-typebarrier is implanted either in conjunction with or independent of thedevice. In a specific embodiment such an optic or contact lens protectsphotoreceptors from light stimulation. In a specific embodiment the lenscomprises tinting over photoreceptors, and clear over light-sensitiveprotein transduction area.

In some embodiments a head-mounted, external device or eyewear isutilized. In certain embodiments where the light-sensitive element isnot triggered to the extent desired by natural or ambient light, anadditional light production or generation source such as a LEDarray/laser system is provided. In certain embodiments the externaleyewear can additionally include a camera and an image processing unitfor the filtering, enhancement, processing, and resolution of thepresented images. FIG. 13 depicts a goggle-like device with anassociated light production element (LED array/laser system) that maytrigger expression of light-sensitive proteins.

In one embodiment, an exemplary camera system would comprise at leastthree main components: 1) A small camera built into the glasses, 2) animaging processing unit, and 3) a light delivery system that includeseither or both LEDs or a laser system. The camera could either be asingle lens camera or a dual camera system that could potentiallyprovide binocular imaging and depth information. The camera couldcapture either visual light or infrared light. The camera could eitherbe adaptive to various lighting conditions or could be fixed. The imageprocessing unit (IPU) could provide any number of signal transformationsincluding amplification, increased or decreased contrast, structure frommotion, edge enhancement, or temporal filtering (i.e., integration).Additionally, saliency algorithms could be employed such that onlycertain objects within the field of view are enhanced (e.g., movingcars, doorways) and less important objects (e.g., clouds), are filteredout. The LED and/or laser lighting array system could contain ahigh-density LED array or a scanning laser system that consists ofeither one (1) or more lasers. The position of the lights could beeither fixed or could move. For example, the orientation of the lightsrelative to the eye could move as a function of eye movements, using aneye movement tracking device as an input. This is depicted in FIG. 13.

In another related exemplary embodiment, an image intensifying device,such as those provided by Telesensory (http://www.telesensory.com), maybe combined with a retinal scanning device (RSD) as developed byMicrovision (http://www.microvision.com/milprod.html), to provide ahead-worn apparatus capable of delivering a bright, intensified imagedirectly to the retina of a patient with impaired vision. Briefly, a RSDprojects images onto the retina such that an individual can view alarge, full-motion image without the need for additional screens ormonitors. Thus, by projecting an intensified image directly onto theretina of an individual with impaired vision, it may be possible toimprove vision in those considered to be blind or near-blind.

While some embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention.

EXAMPLES Example 1 Injection Methods

All procedures in animals were handled according to the statement forthe use of animals in Ophthalmic and Vision Research of the Associationof Research in Vision and Ophthalmology and the guidelines of theInstitutional Animal Care and Use Committee at the University ofFlorida.

For intrvitreal injections, mice were anesthetized with ketamine (72mg/kg)/xylazine (4 mg/kg) by intraperitoneal injection. Followinganesthetization, a Hamilton syringe fitted with a 33-gauge beveledneedle was used. The needle was passed through the sclera, at theequator, next to the limbus, into the vitreous cavity. Injectionoccurred with direct observation of the needle in the center of thevitreous cavity. The total volume delivered was 1.5 containing differentconcentrations of the AAV vectors tested.

For subretinal injections, one hour before the anesthesia, eyes of micewere dilated with eye drops of 1% atropine, followed by topicaladministration of 2.5% phenylephrine. Mice were then anesthetized withketamine (72 mg/kg)/xylazine (4 mg/kg) by intraperitoneal injection Anaperture within the pupil was made through the cornea with a 30½-gaugedisposable needle and a 33-gauge unbeveled blunt needle in a Hamiltonsyringe was introduced through the corneal opening into the subretinalspace and 1.5 μl of AAV was delivered.

Typical titers of the AAV vectors were between 1.3×10¹² and 3.0×10¹³.

Example 2 Screening for AAV Serotypes 1, 2, 5, 7, 8, and 9 forTransduction of Retinal Bipolar Cells

Screening of known and characterized viral vectors for optimaltransduction of retina bipolar cells was carried out. AAV serotypes 1,2, 5, 7, 8 and 9 carrying green fluorescent protein (GFP) wereindividually subretinally injected in 4 week old rd1 mice. Rd1homozygous mice carry a rd1 mutation and rod photoreceptor degenerationin these mice begins around postnatal day (P)10 and is almost completedby P21. GFP was placed under control of the strong, non-cell typespecific promoter CBA (fusion of the CMV immediate early enhancer andthe bovine β-actin promoter plus intron1-exon1 junction). 1 month later,mice were tested for expression of GFP. Double labeling with the PKCαantibody of mice injected with AAV7 demonstrated that the transducedcells were most likely residual (no outer segment) photoreceptors ratherthan bipolar cells. Subretinal injections with AAV7 were then performedin 8 week old mice, where there is less of a chance of residualphotoreceptors. It was found that AAV7 was highly effective attransducing retinal bipolar cells, leading to GFP expression in at least75% of all bipolar cells after a single injection (FIG. 9). These imageswere obtained 16 weeks after injection, which additionally show that GFPexpression using an AAV7 delivery mechanism is stable for at least 4months. AA7 is a serotype that can be utilized to transduce bipolarcells in an effective and stable manner.

Example 3 Transduction of Retinal Bipolar Cells with Serotypes AAV5,AAV2 Y444F Mutant, and AAV8 Y733F Mutant

As depicted in FIG. 10, mice were subretinally (right eye) andintravitreally (left eye) injected with 1.5 μl of adeno-associatedviruses (AAV) of different serotypes. The serotypes tested includedAAV2, AAV5, and AAV8, all of which are traditional wild type serotypes.Additionally, single tyrosine to phenylalanine mutated serotypes AAV2Y444F mutant and AAV8 Y733F mutant, where 444 and 733 indicate thelocation of the point tyrosine mutation of the viral capsid,respectively. The virus contained the self-complementary DNA constructGRM6-ChR2-GFP, where GRM6 is the metabotropic glutamate receptor 6regulatory sequence driving cell-specific expression in the ON bipolarcells (including rod bipolar), ChR2 is the therapeutic, light-sensitiveprotein gene, and GFP is the reporter gene.

The images in FIG. 10 show the overall expression of GFP (the reportergene). This expression is shown as white in the black and white images.This is indicative of the overall expression of ChR2 as the ChR2-GFPcomplex is a fused protein. Note the ringlets of GFP expression in theINL, showing expression of the ChR2-GFP protein complex is membranebound. These data show that delivery of the construct with anadeno-associated virus leads to robust expression of ChR2-GFP in all 3mouse models of blindness (rd1, rd16, and rho −/−). This is conductedwith 3 different serotypes using a subretinal injection (column 1). Whenusing a tyrosine to phenylalanine mutant serotype, it is possible to getgood expression in bipolar cells (INL) using either a subretinal orintravitreal injection. However, wild type serotypes require asubretinal injection to get reasonable transduction of bipolar cells;intravitreal injections using wild type serotypes do not effectivelytransduce bipolar cells (column 2).

Example 4 Creation of AAV7-GRM6-ChR2 to Establish Light Sensitivity inRetinal on-Bipolar Cells

mGluR6 is a G-protein coupled metabotropic glutamate receptor that is,in the retina, specifically expressed in ON bipolar cells (Tian 2006).The adeno-associated virus, serotype 7 (AAV7), an mGluR6 regulatorysequence fragment gene sequence (presented in FIG. 6), GRM6, andchannelrhodopsin-2, ChR2 was constructed to form the AAV7-GRM6-ChR2construct. The cDNA encoding ChR2 with eGFP was cloned downstream (i.e.,3′) of the mGluR6 regulatory sequence fragment-SV40 minimal promoter. NoIRES was used; ChR2 and GFP were fused. This viral vector construct oncedelivered using a viral delivery mechanism, and expressed, can establishphotosensitivity in retinal ON bipolar cells with high-spatial andtemporal resolution. This method can restore retinal responsiveness tooptical information, using the ChR2 class of light-activated moleculesto directly sensitize spared retinal neurons to light.

Example 5 AAV8 Mutant Y733F-GRM6-ChR2 and AAV8 Mutant Y446F-CBA-ChR2 toEstablish Light Sensitivity in Retinal ON-Bipolar Cells

Using a tyrosine-mutated version of AAV8 (at the 733 location), underthe control of bipolar cell specific promoter GRM6, in theself-complementary configuration, it was possible to restore visualbehavioral efficacy in rd1 mice, as depicted in FIGS. 11 and 12. FIG. 11depicts the analysis of EGFP expression in frozen retinal sections byimmunohistochemistry at 1 month following subretinal injections with theTyrosine mutant AAV vectors. Example sections depicting spread andintensity of EGFP fluorescence throughout the retina after transductionwith serotype 2 Y444 (a) or serotype 8 Y733 (b). The images are orientedwith the vitreous toward the bottom and the photoreceptor layer towardthe top. EGFP fluorescence in photoreceptors, RPE and ganglion cellsfrom mouse eyes injected subretinally with serotype 2 Y444 (c) EGFPfluorescence in photoreceptors, RPE and Müller cells after serotype 8Y733 delivery (d) Detection of Müller cells processes (red) byimmunostaining with a glutamine-synthetase (GS) antibody (e) Mergedimage showing colocalization of EGFP fluorescence (green) and GSstaining (red) in retinal sections from eyes treated with serotype 8Y733 (f) Calibration bar 100 μM. gcl, ganglion cell layer; ipl, innerplexiform layer; inl, inner nuclear layer; onl, outer nuclear; os, outersegment; rpe, retinal pigment epithelium.

Using a tyrosine-mutated version of AAV8 (at the 446 location), underthe control of the non-cell specific promoter CBA (fusion of the CMVimmediate early enhancer and the bovine β-actin promoter plusintron1-exon1 junction, and ChR2), in the self-complementaryconfiguration, most or all bipolar cells can be targeted and visualfunction is restored as depicted in FIG. 12.

Example 6 AAV5-CBA-ChR2 to Establish Light Sensitivity in Retinal onBipolar Cells

Using the AAV5, non-cell type specific promoter CBA (fusion of the CMVimmediate early enhancer and the bovine β-actin promoter plusintron1-exon1 junction, and ChR2, in the self-complementaryconfiguration, all bipolar cells can be targeted and visual function andbehavior is restored (FIG. 12). Treated mice were subretinally (righteye) and intravitreally (left eye) injected with 1.5 μl ofadeno-associated viruses (AAV) of different serotypes. The serotypestested included AAV2, AAV5, and AAV7, all of which are traditional wildtype serotypes. Additionally, the single tyrosine to phenylalaninemutated serotypes AAV2 Y444F mutant and AAV8 Y733F mutant, where 444 and733 indicate the location of the point tyrosine mutation of the viralcapsid, respectively. The virus contained the self-complementary DNAconstruct GRM6-ChR2-GFP, where GRM6 is the regulatory sequence drivingcell-specific expression in the ON bipolar cells (including rodbipolar), ChR2 is the therapeutic, light-sensitive protein gene, and GFPis the reporter gene.

These mice were then trained on a water maze task FIG. 12A for 14 days(7 days for the wild type mice) and the time to find the target (a blackplatform with a 4×6 LED light source) was recorded. FIG. 12B shows theaverage time it took for the treated, untreated, and wild type mice tofind the target, as a function of the training session. Both theuntreated and treated groups contained samples from the rd1, rd16, andrho −/− (different mouse models of blindness that have different typesof gene mutations that lead to photoreceptor disease) groups. These datademonstrate that mice treated with ChR2 are able to learn a behaviortask by using visual information, suggesting that a light sensitiveprotein such as ChR2 has the ability to restore at least some visualfunction.

The animals' performance on the task was then evaluated at differentlight levels. FIG. 12C shows the average time it took for the rd1, rd16,and rho −/− treated, sham injected (sham injected mice represent anaverage of rd1, rd16, and rho −/− untreated), and wild type mice to findthe target, as a function of the light intensity. These data show thatthe treated mice can perform the task at multiple light levels and theirperformance is dependent on the amount of light presented.

What is claimed is:
 1. A recombinant nucleic acid comprising a nucleicacid encoding a light-sensitive protein operatively linked to ametabotropic glutamate receptor 6 (mGluR6) regulatory sequence orfragment thereof.
 2. The nucleic acid of claim 1 wherein thelight-sensitive protein is selected from the group consisting of ChR1,ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, andvariants thereof.
 3. The nucleic acid of claim 1 wherein thelight-sensitive protein is ChR2 or a light-sensitive protein that is atleast about 70%, at least about 80%, at least about 90% or at leastabout 95% identical to ChR2.
 4. The nucleic acid of claim 1 wherein themGluR6 regulatory sequence fragment comprises less than about 1000, lessthan about 750, less than about 500, less than about 250, or less thanabout 100 base pairs.
 5. The nucleic acid of claim 1 wherein the mGluR6regulatory sequence or fragment thereof is an mGluR6 promoter orenhancer.
 6. The nucleic acid of claim 1 further comprising a greenfluorescent protein
 7. The nucleic acid of claim 1 wherein the nucleicacid is encapsidated within a recombinant adeno-associated virus (AAV)8. The nucleic acid of claim 7 wherein the recombinant adeno-associatedvirus is of a serotype selected from the group consisting of AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, andhybrids thereof.
 9. The nucleic acid of claim 7 wherein the recombinantadeno-associated virus is of a serotype selected from the groupconsisting of AAV2, AAV5, AAV7, AAV8, and hybrids thereof.
 10. Thenucleic acid of claim 1 wherein the nucleic acid is encapsidated withina recombinant virus selected from the group consisting of recombinantadeno-associated virus (AAV), recombinant retrovirus, recombinantlentivirus, and recombinant poxvirus.
 11. A vector comprising a nucleicacid encoding a light-sensitive protein, said nucleic acid operativelylinked to a metabotropic glutamate receptor 6 (mGluR6) regulatorysequence or fragment thereof.
 12. The vector of claim 11 wherein thelight-sensitive protein is selected from the group consisting of ChR1,ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, andvariants thereof.
 13. The nucleic acid of claim 11, wherein thelight-sensitive protein is ChR2 or a light-sensitive protein that is atleast about 70%, at least about 80%, at least about 90% or at leastabout 95% identical to ChR2.
 14. The vector of claim 11 wherein themGluR6 regulatory sequence fragment is less than about 1000, less thanabout 750, less than about 500, less than about 250, or less than about100 base pairs.
 15. The vector of claim 11 wherein the mGluR6 regulatorysequence fragment is represented by the sequence in FIG.
 6. 16. Thevector of claim 11 wherein the vector comprises a recombinantadeno-associated virus (AAV).
 17. The vector of claim 11 wherein thevector comprises a recombinant virus selected from the group consistingof recombinant adeno-associated virus (AAV), recombinant retrovirus,recombinant lentivirus, and recombinant poxvirus.
 18. The vector ofclaim 16 wherein the AAV is of a serotype selected from the groupconsisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAV10, AAV11, AAV12, and hybrids thereof.
 19. The vector of claim 18wherein the AAV comprises mutated capsid protein.
 20. The vector ofclaim 19 wherein the capsid protein comprises a mutated tyrosineresidue.
 21. The vector of claim 20 wherein the mutated tyrosine residueis selected from the group consisting of Y252F, Y272F, Y444F, Y500F,Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and Y720F.22. The vector of claim 20 wherein the mutated capsid protein comprisesa tyrosine residue mutated to a phenylalanine.
 23. A method of treatinga subject suffering from a disease or disorder of the eye comprisingintroducing into an affected eye a recombinant adeno-associated virus(AAV) comprising a light-sensitive protein operatively linked to ametabotropic glutamate receptor 6 regulatory sequence (mGluR6 regulatorysequence) or fragment thereof.
 24. The method of claim 23 wherein thedisease or disorder of the eye is caused by photoreceptor celldegeneration.
 25. The method of claim 23 wherein the light-sensitiveprotein is selected from the group consisting of ChR1, ChR2, VChR1, ChR2C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF,ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants thereof.
 26. Themethod of claim 23 wherein the light-sensitive protein is ChR2 or alight-sensitive protein that is at least about 70%, at least about 80%,at least about 90% or at least about 95% identical to ChR2.
 27. Themethod of claim 23 wherein the mGluR6 regulatory sequence fragment isless than about 1000, less than about 750, less than about 500, lessthan about 250, or less than about 100 base pairs.
 28. The method ofclaim 27 wherein the mGluR6 regulatory sequence fragment is representedby the sequence in FIG.
 6. 29. The method of claim 23 wherein the AAV isof a serotype selected from the group consisting of AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybridsthereof.
 30. The method of claim 29 wherein the AAV comprises a mutatedcapsid protein.
 31. The method of claim 30 wherein the capsid proteincomprises a mutated tyrosine residue.
 32. The method of claim 31 whereinthe mutated tyrosine residue is selected from the group consisting ofY252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F,Y576F, Y612G, Y673F and Y720F.
 33. The vector of claim 31 wherein themutated capsid protein comprises a tyrosine residue mutated to aphenylalanine.
 34. The method of claim 23 wherein the AAV is introducedusing intravitreal injection, subretinal injection and/or ILM peel. 35.The method of claim 23 wherein the AAV is introduced into a retinalbipolar cell.
 36. The method of claim 23 wherein the method furthercomprises using a light-generating device external to the eye.
 37. Amethod of expressing an exogenous nucleic acid in a retinal bipolar cellcomprising introducing into a retina a vector comprising the exogenousnucleic operatively linked to a retinal bipolar cell-specific regulatorysequence wherein the method results in at least about a 25-30%transduction efficiency.
 38. The method of claim 36 wherein the methodresults in at least about a 40%, 50%, 60%, 70%, 80%, or 90% transductionefficiency.
 39. The method of claim 36 wherein the transductionefficiency is measured by quantifying the total number of retinalbipolar cells infected.
 40. The method of claim 36 wherein the exogenousnucleic acid comprises a light-sensitive protein.
 41. The method ofclaim 40 wherein the light-sensitive protein is selected from the groupconsisting of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T,ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR,melanopsin, and variants thereof.
 42. The nucleic acid of claim 40wherein the light-sensitive protein is ChR2 or a light-sensitive proteinthat is at least about 70%, at least about 80%, at least about 90% or atleast about 95% identical to ChR2.
 43. The method of claim 36 whereinthe regulatory sequence comprises a metabotropic glutamate receptor 6regulatory sequence (mGluR6) or a fragment thereof.
 44. The method ofclaim 43 wherein the mGluR6 regulatory sequence fragment is less thanabout 1000, less than about 750, less than about 500, less than about250, or less than about 100 base pairs.
 45. The method of claim 43wherein the mGluR6 regulatory sequence fragment is represented by thesequence in FIG.
 6. 46. The method of claim 36 wherein the exogenousnucleic acid is introduced using a recombinant adeno-associated viralvector (AAV).
 47. The method of claim 46 wherein the AAV comprises amutated capsid protein.
 48. The method of claim 47 wherein the capsidprotein comprises a mutated tyrosine residue.
 49. The method of claim 48wherein the mutated tyrosine residue is selected from the groupconsisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F,Y281F, Y508F, Y576F, Y612G, Y673F and Y720F.
 50. The vector of claim 48wherein the mutated capsid protein comprises a tyrosine residue mutatedto a phenylalanine.
 51. The method of claim 46 wherein the exogenousnucleic acid is introduced using intravitreal injection, subretinalinjection, and/or ILM peel.
 52. The method of claim 46 wherein the AAVis of a serotype is selected from the group consisting of AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, andhybrids thereof.
 53. A method of introducing an exogenous nucleic acidinto the nucleus of a retinal cell comprising introducing a vectorcomprising an exogenous nucleic acid operatively linked to a retinalcell-specific regulatory sequence into a retinal cell, wherein thevector is specifically designed to avoid ubiquitin-mediated proteindegradation.
 54. The method of claim 53 wherein the degradation isproteasome-mediated.
 55. The method of claim 53 wherein the exogenousnucleic acid comprises a light-sensitive protein.
 56. The method ofclaim 55 wherein the light-sensitive protein is selected from the groupconsisting of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T,ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR,melanopsin, and variants thereof.
 57. The nucleic acid of claim 55wherein the light-sensitive protein is ChR2 or a light-sensitive proteinthat is at least about 70%, at least about 80%, at least about 90% or atleast about 95% identical to ChR2.
 58. The method of claim 53 whereinthe retinal cell is a retinal bipolar cell.
 59. The method of claim 58wherein the regulatory sequence comprises a metabotropic glutamatereceptor 6 regulatory sequence (mGluR6) or fragment thereof.
 60. Themethod of claim 59 wherein the mGluR6 fragment is less than 1000, 750,500, 250, or 100 base pairs.
 61. The method of claim 59 wherein themGluR6 regulatory sequence fragment is represented by the sequence inFIG.
 6. 62. The method of claim 53 wherein the vector is selected fromthe group consisting of recombinant adeno-associated virus (AAV),recombinant retrovirus, recombinant lentivirus, and recombinantpoxvirus.
 63. The method of claim 53 the vector is a recombinantadeno-associated viral vector (AAV).
 64. The method of claim 63 whereinthe AAV is of a serotype selected from the group consisting of AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, andhybrids thereof.
 65. The method of claim 63 wherein the AAV comprises amutated capsid protein.
 66. The method of claim 65 wherein the capsidprotein comprises a mutated tyrosine residue.
 67. The method of claim 66wherein the mutated tyrosine residue is selected from the groupconsisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F,Y281F, Y508F, Y576F, Y612G, Y673F and Y720F.
 68. The vector of claim 66wherein the mutated capsid protein comprises a tyrosine residue mutatedto a phenylalanine.
 69. The method of claim 53 wherein the vector isintroduced using intravitreal injection, subretinal injection, and/orILM peel.
 70. A method of transducing a retinal bipolar cell comprisingintroducing into a retina a vector comprising an exogenous nucleic acidoperatively linked to a regulatory sequence.
 71. The method of claim 70wherein the regulatory sequence is a non-cell type specific promoter.72. The method of claim 70, wherein the regulatory sequence is a guaninenucleotide binding protein alpha activating activity polypeptide O(GNAO1) promoter or a fusion of the cytomegalovirus (CMV) immediateearly enhancer and the bovine β-actin promoter plus intron1-exon1junction (CBA, smCBA).
 73. The method of claim 70 wherein the exogenousnucleic acid comprises a light-sensitive protein.
 74. The method ofclaim 73 wherein the light-sensitive protein is selected from the groupconsisting of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T,ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR,melanopsin, and variants thereof.
 75. The nucleic acid of claim 73wherein the light-sensitive protein is ChR2 or a light-sensitive proteinthat is at least about 70%, at least about 80%, at least about 90% or atleast about 95% identical to ChR2.
 76. The method of claim 70 whereinthe vector is selected from the group consisting of recombinantadeno-associated virus (AAV), recombinant retrovirus, recombinantlentivirus, and recombinant poxvirus.
 77. The method of claim 70 thevector is a recombinant adeno-associated viral vector (AAV).
 78. Themethod of 77 wherein the AAV is of a serotype selected from the groupconsisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAV10, AAV11, AAV12, and hybrids thereof.
 79. The method of 77 whereinthe AAV comprises a mutated capsid protein.
 80. The method of claim 79wherein the capsid protein comprises a mutated tyrosine residue.
 81. Themethod of claim 79 wherein the mutated tyrosine residue is selected fromthe group consisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F,Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and Y720F.
 82. The vector ofclaim 80 wherein the mutated capsid protein comprises a tyrosine residuemutated to a phenylalanine.
 83. The method of claim 70 wherein thevector is introduced using intravitreal injection, subretinal injection,and/or ILM peel.